IL-8 Like Protein

ABSTRACT

This present invention relates to a novel protein, termed INSP085, herein identified as an IL-8 like protein and to the use of this protein and nucleic acid sequence from the encoding genes in the diagnosis, prevention and treatment of disease.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International PatentApplication PCT/GB2003/003646 filed Aug. 19, 2003 and published as WO2004/016654 on Feb. 26, 2004, which claims priority from Great BritainApplication 0219303.5 filed Aug. 19, 2002. Each of the above referencedapplications, and each document cited in this text (“application citeddocuments”) and each document cited or referenced in each of theapplication cited documents, and any manufacturer's specifications orinstructions for any products mentioned in this text and in any documentincorporated into this text, are hereby incorporated herein byreference; and, technology in each of the documents incorporated hereinby reference can be used in the practice of this invention.

It is noted that in this disclosure, terms such as “comprises”,“comprised”, “comprising”, “contains”, “containing” and the like canhave the meaning attributed to them in U.S. patent law; e.g., they canmean “includes”, “included”, “including” and the like. Terms such as“consisting essentially of” and “consists essentially of” have themeaning attributed to them in U.S. patent law, e.g., they allow for theinclusion of additional ingredients or steps that do not detract fromthe novel or basic characteristics of the invention, i.e., they excludeadditional unrecited ingredients or steps that detract from novel orbasic characteristics of the invention, and they exclude ingredients orsteps of the prior art, such as documents in the art that are citedherein or are incorporated by reference herein, especially as it is agoal of this document to define embodiments that are patentable, e.g.,novel, nonobvious, inventive, over the prior art, e.g., over documentscited herein or incorporated by reference herein. And, the terms“consists of” and “consisting of” have the meaning ascribed to them inU.S. patent law; namely, that these terms are closed ended.

This invention relates to a novel protein, termed INSP085, hereinidentified as a secreted protein, in particular, as a member of theInterleukin (IL) 8-like chemokine family and to the use of this proteinand nucleic acid sequence from the encoding gene in the diagnosis,prevention and treatment of disease.

All publications, patents and patent applications cited herein areincorporated in full by reference.

BACKGROUND

The process of drug discovery is presently undergoing a fundamentalrevolution as the era of functional genomics comes of age. The term“functional genomics” applies to as approach utilising bioinformaticstools to ascribe function to protein sequences of interest. Such toolsare becoming increasingly necessary as the speed of generation ofsequence data is rapidly outpacing the ability of research laboratoriesto assign functions to these protein sequences.

As bioinformatics tools increase in potency and in accuracy, these toolsare rapidly replacing the conventional techniques of biochemicalcharacterisation. Indeed, the advanced bioinformatics tools used inidentifying the present invention are now capable of outputting resultsin which a high degree of confidence can be placed.

Various institutions and commercial organizations are examining sequencedata as they become available and significant discoveries are being madeon an on-going basis. However, there remains a continuing need toidentify and characterise further genes and the polypeptides that theyencode, as targets for research and for drug discovery.

INTRODUCTION Secreted Proteins

The ability for cells to make and secrete extracellular proteins iscentral to many biological processes. Enzymes, growth factors,extracellular matrix proteins and signalling molecules are all secretedby cells. This is through fission of a secretory vesicle with theplasmas membrane. In most cases, but not all, proteins are directed tothe endoplasmic reticulum and into secretory vesicles by a signalpeptide. Signal peptides are cis-acting sequences that affect thetransport of polypeptide chains from the cytoplasm to a membrane boundcompartment such as a secretory vesicle. Polypeptides that are targetedto the secretory vesicles are either secreted into the extracellularmatrix or are retained in the plasma membrane. The polypeptides that areretained in the plasma membrane will have one or more transmembranedomains. Examples of secreted proteins that play a central role in thefunctioning of a cell are cytokines, hormones, extracellular matrixproteins (adhesion molecules), proteases, and growth and differentiationfactors. Description of some of the properties of these proteinsfollows.

Chemokines

These signalling molecules are distinct from cytokines and areresponsible for inducing chemotaxis or directed migration. They arehighly specific, a fact which is illustrated by the fact that IL-8 ischemotactic to granulocytes but not monocytes. Chemokines contain fourconserved cysteine residues and are divided into three families, α(CXC),β(CC) and γ(C), based on the position of conserved cysteine residues. Ifthe first two cysteines are separated by another amino acid, then thechemokine is a member of the α family, while the first two cysteineresidues are next to each other in the β family members. Members of theγ family only have one cysteine residue, rather than two, in theirN-terminus. In the α and β families, disulphide bonds are formed betweenthe first and third and the second and fourth residues.

Specificity of chemokines depends on the presence of specific receptorson cell surfaces. Chemokines have been shown to play a role in themigration of leukocytes. Upon activation, remodeling of the cytoskeletonof leukocytes is induced allowing the cell to flatten and pass from anintravascular space into a tissue space. Interaction of chemokines withseven-transmembrane G-protein coupled receptors leads to rapidaccumulation of intracellular free calcium in the responding cells. Thismobilization is critical for chemotaxis, respiratory burst andupregulation of adhesive interactions of leukocytes. Chemokines havealso been shown to regulate the expression of adhesion molecules onneutrophils, monocytes, lymphocytes and eosinophils. For example, MIP-1αand RANTES cause adhesion of monocytes to endothelium while MIP-1βinduces CD8⁺ T-cell adhesion to endothelium.

Increasing knowledge of these domains is therefore of extreme importancein increasing the understanding of the underlying pathways that lead tothe disease states and associated disease states mentioned above, and indeveloping more effective gene and/or drug therapies to treat thesedisorders.

THE INVENTION

The invention is based on the discovery that the INSP085 polypeptide isan IL-8 like chemokine.

In one embodiment of the first aspect of the invention, there isprovided a polypeptide which:

-   (i) comprises the amino acid sequence as recited in SEQ ID NO:2, SEQ    ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO10 and/or SEQ ID NO:12;-   (ii) is a fragment thereof which functions as a member IL-8 like    chemokine family, or has an antigenic determinant in common with the    polypeptides of (i); or-   (iii) is a functional equivalent of (i) or (ii).

Preferably, the polypeptide according to this first aspect of theinvention:

-   (i) comprises the amino acid sequence as recited in SEQ ID NO:8, SEQ    ID NO:10 and/or SEQ ID NO:12;-   (ii) is a fragment thereof which functions as a member of the IL-8    like chemokine family, or has an antigenic determinant in common    with the polypeptides of (i); or-   (iii) is a functional equivalent of (i) or (ii).

According to a second embodiment of this first aspect of the invention,there is provided a polypeptide which:

-   (i) consists of the amino acid sequence as recited in SEQ ID NO:2,    SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and/or SEQ ID    NO:12;-   (ii) is a fragment thereof which functions as a member of the IL-8    like chemokine family, or having an antigenic determinant in common    with the polypeptides of (i); or-   (iii) is a functional equivalent of (i) or (ii).

The polypeptide having the sequence recited in SEQ ID NO:2 is referredto hereafter as “INSP085 exon 1 polypeptide”. The polypeptide having thesequence recited in SEQ ID NO:4 is referred to hereafter as “INSP085exon 2 polypeptide”. The polypeptide having the sequence recited in SEQID NO:6 is referred to hereafter as “INSP085 exon 3 polypeptide”. Thepolypeptide having the sequence recited in SEQ ID NO:8 is referred tohereafter as the “INSP085 polypeptide”.

The term “INSP085 polypeptides” as used herein includes polypeptidescomprising the INSP085 exon 1 polypeptide, the INSP085 exon 2polypeptide, the INSP085 exon 3 polypeptide and the NSP085 polypeptide.

Although the Applicant does not wish to be bound by this theory, it ispostulated that the first 16 amino acids of the INSP085 exon 1polypeptide form a signal peptide.

The INSP085 exon 1 and full length INSP085 polypeptide sequences withoutthis postulated signal sequence are recited in SEQ ID NO:10 and SEQ IDNO:12 respectively.

The polypeptide having the sequence recited in SEQ ID NO:10 is referredto hereafter as “the INSP085 exon 1 mature polypeptide”. The polypeptidehaving the sequence recited in SEQ ID NO:12 is referred to hereafter as“the INSP085 mature polypeptide”.

By “functions as a member of the IL 8 like chemokine family” we refer topolypeptides that comprise amino acid sequence or structural featuresthat can be identified as conserved features within the polypeptides ofthe IL-8 like chemokine family, such that the polypeptide's interactionwith ligand is not substantially affected detrimentally in comparison tothe function of the full length wild type polypeptide. In particular, werefer to the presence of cysteine residues in specific positions withinthe polypeptide that allow the formation of intra-domain disulphidebonds.

Studies on structure-activity relationships indicate that chemokinesbind and activate receptors by making use of the amino-terminal region.Proteolytic digestion, mutagenesis, or chemical modifications directedto amino acids in this region can generate compounds having antagonisticactivity (Loetscher P and Clark-Lewis I, J Leukoc Biol, 69: 881-884,2001 Lambeir A, et al. J Biol Chem, 276: 29839-29845, 2001, Proost P, etal. Blood, 98 (13):3554-3561, 2001). Thus, antagonistic moleculesresulting from specific modifications (deletions, non-conservativesubstitutions) of on-e or more residues in the amino-terminal region orin other regions of the corresponding chemokine are considered havingtherapeutic potential for inflammatory and autoimmune diseases (WO02/28419; WO 00/27880; WO 99/33989; Schwarz M K and Wells T N, Curr OpinChem Biol, 3: 407-17, 1999). Therefore, a further object of the presentpatent application is represented by such kind of antagonists generatedby modifying the polypeptides of the invention.

The therapeutic applications of the polypeptides of the invention and ofthe related reagents can be evaluated (in terms of safety,pharmacokinetics and efficacy) by the means of the in vivo/in vitroassays making use of animal cell, tissues and models (Coleman R A etal., Drug Discov Today, 6: 1116-1126, 2001; Li AP, Drug Discov Today, 6:357-366, 2001; Methods Mol. Biol. vol. 138, “Chemokines Protocols”,edited by Proudfoot A I et al., Humana Press Inc., 2000; MethodsEnzymol, vol. 287 and 288, Academic Press, 1997), or by the means of insilico/computational approaches (Johnson D E and Wolfgang G H, DrugDiscov Today, 5: 445-454, 2000), known for the validation of chemokinesand other biological products during drug discovery and preclinicaldevelopment.

The present application discloses novel chemokine-like polypeptides ad aseries of related reagents that may be useful, as active ingredients inpharmaceutical compositions appropriately formulated, in the treatmentor prevention of diseases such as cell proliferative disorders,autoimmune/inflammatory disorders, cardiovascular disorders,neurological disorders, developmental disorders, metabolic disorder,infections and other pathological conditions. In particular, given theknown properties of chemokines, the disclosed polypeptides and reagentsshould address conditions involving abnormal or defective cellmigration. Non-limitative examples of such conditions are the following:arthritis, rheumatoid arthritis (RA), psoriatic arthritis,osteoarthritis, systemic lupus erythematosus (SLE), systemic sclerosis,scleroderma, polymyositis, glomerulonephritis, fibrosis, lung fibrosisand inflammation, allergic or hypersensitivity diseases, dermatitis,asthma, chronic obstructive pulmonary disease (COPD), inflammatory boweldisease (IBD), Crohn's disease, ulcerative colitis, multiple sclerosis,septic shock, HIV infection, transplant rejection, wound healing,metastasis, endometriosis, hepatitis, liver fibrosis, cancer, analgesia,and vascular inflammation related to atherosclerosis.

Several assays have been developed for testing specificity, potency, andefficacy of chemokines using cell cultures or animal models, for examplein vitro chemotaxis assays (Proudfoot A, et al. J Biol Chem 276:10620-10626, 2001; Lusti-Naurasimhan M et al., J Biol Chem, 270:2716-21, 1995), or mouse ear swelling (Garrigue J L et al., ContactDermatitis, 30: 231-7, 1994). Many other assays and technologies forgenerating useful tools and products (antibodies, transgenic animals,radiolabeled proteins, etc.) have been described in reviews and booksdedicated to chemokines (Methods Mol. Biol vol. 138, “ChemokinesProtocols”, edited by Proudfoot A I et al., Humana Press Inc., 2000;Methods Enzymol, vol. 287 and 288, Academic Press, 1997), and can beused to verify, in a more precise manner, the biological activities ofthe chemokine-like polypeptides of the invention and related reagents inconnection with possible therapeutic or diagnostic methods and uses.

The following in vitro cell-based tri-replicas assays measure theeffects of the protein of the invention on cytokine secretion induced byConcanavalin A (Con A) acting on different human peripheral bloodmononuclear cells (hPBMC) cells as measured by a cytokine bead array(CBA) assay for IL-2, IFN-γ, TNF-α, IL-5, IL-4 and IL-10 such as theHuman Th1/Th2 Cytokine CBA kit (Becton-Dickinson).

The optimal conditions are 100000 cells/well in 96-well plates and 100μl final in 2% glycerol. The optimal concentration of mitogen (ConA) is5 ng/ml. The optimal time for the assay is 48 h. The read-out choice isthe CBA.

1 Purification of Human PBMC from a Buffy Coat

The buffy coat 1 to 2 is diluted with DMEM. 25 ml of diluted blood wasthereafter slowly added onto a 15 ml layer of Ficoll in a 50 ml Falcontube, and tubes are centrifuged (2000 rpm, 20 min, at RT without brake).The interphase (ring) is then collected and the cells are washed with 25ml of DMEM followed by a centrifuge step (1200 rpm, 5 min). Thisprocedure is repeated three times. A but coat gives approximately600×10⁶ total cells.

2 Screening

80 μl of 1.25×10⁶ cells/ml are diluted in DMEM+2.5% Human Serum+1%L-Glutamine+1% Penicillin-Streptomycin and thereafter added to a 96 wellmicrotiter plate.

10 μl are added per well (one condition per well): Proteins were dilutedin PBS+20% Glycerol (the final dilution of the proteins is 1/10).

10 μl of the ConA Stimulant (50 μg/ml) are then added per well (onecondition per well—the final concentration of ConA is 5 μg/ml)

After 48 h, cell supernatants are collected and human cytokines aremeasured by Human Th1/Th2 Cytokine CBA Kit Becton-Dickinson.

3 CBA Analysis

(for more details, refer to the manufacturer's instructions in the CBAkit)i) Preparation of mixed Human Th1/Th2 Capture Beads

The number of assay tubes that are required for the experiment aredetermined.

Each capture bead suspension is vigorously vortexed for a few secondsbefore mixing. For each assay to be analysed, 10 μl aliquot of eachcapture bead are added into a single tube labelled “mixed capturebeads”. The Bead mixture is thoroughly vortexed.

ii) Preparation of Test Samples

Supernatants are diluted (1:4) using the Assay Diluent (20 μl ofsupernatants+60 μl of Assay Diluent) The sample dilution is then mixedbefore transferring samples into a 96 well conical bottomed microtiterplate (Nunc).

iii) Human Th1/Th2 Cytokine CBA Assay Procedure

50 μl of the diluted supernatants are added into a 96 well conicalbottomed microtiter plate (Nunc). 50 μl of the mixed capture beads areadded followed by 50 μl addition of the Human Th1/Th2 PE DetectionReagent. The plate is then incubated for 3 hours at RT and protectedfrom direct exposure to light followed by centrifugation at 1500 rpm for5 minutes. The supernatant is then carefully discarded. In a subsequentstep, 200 μl of wash buffer are twice added to each well, centrifuged at1500 rpm for 5 minutes and supernatant carefully discarded. 130 μl ofwash buffer are thereafter added to each well to resuspended the beadpellet. The samples are finally analysed on a flow cytometer. The dataare then analysed using the CBA Application Software, Activity Base andMicrosoft Excel software.

From the read-out of the assay it can be evaluated whether in vitro, theprotein of the invention has a consistent inhibitory effect on allcytokines tested (IFN-γ, TNF-α, IL-2, IL-4, IL-S, IL-10).

Moreover, based on the EC50 value, it can be easily evaluated whichcytokine is inhibited the most and then derive the specificauto-immune/inflammatory disease, which is known to be particularlylinked to that cytokine.

In a second aspect, the invention provides a purified nucleic acidmolecule which encodes a polypeptide of the first aspect of theinvention.

Preferably, the purified nucleic acid molecule comprises the nucleicacid sequence as recited in SEQ ID NO:1 (encoding the INSP085 exon 1polypeptide), SEQ ID NO:3 (encoding the INSP085 exon 2 polypeptide), SEQID NO:5 (encoding the INSP085 exon 3 polypeptide) and/or SEQ ID NO:7(encoding the INSP085 polypeptide) or is a redundant equivalent orfragment of any one of these sequences.

The invention further provides that the purified nucleic acid moleculeconsists of the nucleic acid sequence as recited in SEQ ID NO:1(encoding the INSP085 exon 1 polypeptide), SEQ ID NO:3 (encoding theINSP085 exon 2 polypeptide), SEQ ID NO:5 (encoding the INSP085 exon 3polypeptide) and/or SEQ ID NO:7 (encoding the INSP085 polypeptide) or isa redundant equivalent or fragment of any one of these sequences.

The polypeptide having the sequence recited in SEQ ID NO:9 is referredto hereafter as “the INSP085 exon 1 mature nucleotide sequence” andencodes the INSP085 exon 1 mature polypeptide. The polypeptide havingthe sequence recited in SEQ ID NO:11 is referred to hereafter as “theINSP08-5 mature nucleotide sequence” and encodes the INSP085 maturepolypeptide.

In a third aspect, the invention provides a purified nucleic acidsmolecule which hybridizes under high stringency conditions with anucleic acid molecule of the second aspect of the invention.

In a fourth aspect, the invention provides a vector, such as anexpression vector, that contains a nucleic acid molecule of the secondor third aspect of the invention.

In a fifth aspect, the invention provides a host cell transformed with avector of the fourth aspect of the invention.

In a sixth aspect, the invention provides a ligand which bindsspecifically to protein members of the IL-8 like chemokine family of thefirst aspect of the invention. Preferably, the ligand inhibits thefunction of a polypeptide of the first aspect of the invention which isa member of the IL-8 like chemokine family or proteins Ligands to apolypeptide according to the invention may come in various forms,including natural or modified substrates, enzymes, receptors, smallorganic molecules such as small natural or synthetic organic moleculesof up to 2000 Da, preferably 800 Da or less, peptidomimetics, inorganicmolecules, peptides, polypeptides, antibodies, structural or functionalmimetics of the aforementioned.

In a seventh aspect, the invention provides a compound that is effectiveto alter the expression of a natural gene which encodes a polypeptide ofthe first aspect of the invention or to regulate the activity of apolypeptide of the first aspect of the invention.

A compound of the seventh aspect of the invention Hay either increase(agonize) or decrease (antagonize) the level of expression of the geneor the activity of the polypeptide.

Importantly, the identification of the function of the INSP085polypeptides allows for the design of screening methods capable ofidentifying compounds that are effective in the treatment and/ordiagnosis of disease. Ligands and compounds according to the sixth andseventh aspects of the invention may be identified using such methods.These methods are included as aspects of the present invention.

In an eighth aspect, the invention provides a polypeptide of the firstaspect of the invention, or a nucleic acid molecule of the second orbird aspect of the invention, or a vector of the fourth aspect of theinvention, or a host cell of the fifth aspect of the invention, or aligand of the sixth aspect of the invention, or a compound of theseventh aspect of the invention, for use in therapy or diagnosis ofdiseases in which members of the IL-8 like chemokine family areimplicated. Such diseases may include cell proliferative disorders,including neoplasm, melanoma, lung, colorectal, breast, pancreas, headand neck and other solid tumours; myeloproliferative disorders, such asleukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia,angiogenesis disorder, Kaposis' sarcoma, autoimmune/inflammatorydisorders, including allergy, inflammatory bowel disease, arthritis,psoriasis and respiratory tract inflammation, asthma, and organtransplant rejection; cardiovascular disorders, including hypertension,oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusioninjury, and ischemia; neurological disorders including central nervoussystem disease, Alzheimer's disease, brain injury, amyotrophic lateralsclerosis, and pain, developmental disorders; metabolic disordersincluding diabetes mellitus, osteoporosis, and obesity, AIDS and renaldisease; infections including viral infection, bacterial infection,fungal infection and parasitic infection and other pathologicalconditions. Preferably, the disease is one in which the IL-8 likechemokine family is implicated, such as arthritis, rheumatoid arthritis(RA), psoriatic arthritis, osteoarthritis, systemic lupus erythematosus(SLE), systemic sclerosis, scleroderma, polymyositis,glomerulonephritis, fibrosis, lung fibrosis and inflammation, allergicor hypersensitivity diseases, dermatitis, asthma, chronic obstructivepulmonary disease (COPD), inflammatory bowel disease (IBD), Crohn'sdiseases, ulcerative colitis, multiple sclerosis, septic shock, HIVinfection, transplant rejection, wound healing, metastasis,endometriosis, hepatitis, liver fibrosis, cancer, analgesia, andvascular inflammation related to atherosclerosis. These molecules mayalso be used in the manufacture of a medicament for the treatment ofsuch diseases. These molecules may also be used in contraception or forthe treatment of reproductive disorders including infertility.

In a ninth aspect, the invention provides a method of diagnosing adisease in a patient, comprising assessing the level of expression of anatural gene encoding a polypeptide of the first aspect of the inventionor the activity of a polypeptide of the first aspect of the invention intissue from said patient and comparing said level of expression oractivity to a control level, wherein a level that is different to saidcontrol level is indicative of disease. Such a method will preferably becarried out in vitro. Similar methods may be used for monitoring thetherapeutic treatment of disease in a patient, wherein altering thelevel of expression or activity of a polypeptide or nucleic acidmolecule over the period of time towards a control level is indicativeof regression of disease.

A preferred method for detecting polypeptides of the first aspect of theinvention comprises the steps of: (a) contacting a ligand, such as anantibody, of the sixth aspect of the invention with a biological sampleunder conditions suitable for the formation of a ligand-polypeptidecomplex; and (b) detecting said complex.

A number of different such methods according to the ninth aspect of theinvention exist, as the skilled reader will be aware, such as methods ofnucleic acid hybridization with short probes, point mutation analysis,polymerase chain reaction (PCR) amplification and methods usingantibodies to detect aberrant protein levels. Similar methods may beused on a short or long term basis to allow therapeutic treatment of adisease to be monitored in a patient. The invention also provides kitsthat are useful in these methods for diagnosing disease.

In a tenth aspect, the invention provides for the use of a polypeptideof the first aspect of the invention as an IL-8 like chemokine. Suitableuses of the polypeptides of the invention as IL-8 like chemokineproteins include use as a regulator of cellular growth, metabolism ordifferentiation, use as part of a receptor/ligand pair and use as adiagnostic marker for a physiological or pathological condition selectedfrom the list given above.

In an eleventh aspect, the invention provides a pharmaceuticalcomposition comprising a polypeptide of the first aspect of theinvention, or a nucleic acid molecule of the second or third aspect ofthe invention, or a vector of the fourth aspect of the invention, or ahost cell of the fifth aspect of the invention, or a ligand of the sixthaspect of the invention, or a compound of the seventh aspect of theinvention, in conjunction with a pharmaceutically-acceptable carrier.

In a twelfth aspect, the present invention provides a polypeptide of thefirst aspect of the invention, or a nucleic acid molecule of the secondor third aspect of the inventions or a vector of the fourth aspect ofthe invention, or a host cell of the fifth aspect of the invention, or aligand of the sixth aspect of the invention, or a compound of theseventh aspect of the invention, for use in the manufacture of amedicament for the diagnosis or treatment of a disease.

In a thirteenth aspect, the invention provides a method of treating adisease in a patient comprising administering to the patient apolypeptide of the first aspect of the invention, or a nucleic acidmolecule of the second or third aspect of the invention, or a vector ofthe fourth aspect of the invention, or a host cell of the fifth aspectof the invention, or a ligand of the sixth aspect of the invention, or acompound of the seventh aspect of the invention.

For diseases in which the expression of a natural gene encoding apolypeptide of the first aspect of the invention, or in which theactivity of a polypeptide of the first aspect of the invention, is lowerin a diseased patient when compared to the level of expression oractivity in a healthy patient, the polypeptide, nucleic acid molecule,ligand or compound administered to the patient should be an agonist.Conversely, for diseases in which the expression of the natural gene oractivity of the polypeptide is higher in a diseased patient whencompared to the level of expression or activity in a healthy patient,the polypeptide, nucleic acid molecule, ligand or compound administeredto the patient should be an antagonist Examples of such antagonistsinclude antisense nucleic acid molecules, ribozymes and ligands, such asantibodies.

In a fourteenth aspect, the invention provides transgenic or knockoutnon-human animals that have been transformed to express higher, lower orabsent Levels of a polypeptide of the first aspect of the invention.Such transgenic animals are very useful models for the study of diseaseand may also be used in screening requires for the identification ofcompounds that are effective in the treatment or diagnosis of such adisease.

A summary of standard techniques and procedures which may be employed inorder to utilise the invention is given below. It will be understoodthat this invention is not limited to the particular methodology,protocols, cell lines, vectors and reagents described. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and it is not intended that thisterminology should limit the scope of the present invention. The extentof the invention is limited only by the terms of the appended claims.

Standard abbreviations for nucleotides and amino acids are used in thisspecification.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology and immunology, which are within the skill ofthose working in the art.

Such techniques are explained fully in the literature. Examples ofparticularly suitable texts for consultation include the following:Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989);DNA Cloning, Volumes I and II (D. N Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames &S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986);Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide to Molecular Cloning (1984); the Methods in Enzymology series(Academic Press, Inc.), especially volumes 154 & 155; Gene TransferVectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory); Immunochemical Methods in Cell andMolecular Biology (Mayer and Walker, eds. 1987, Academic Press, London);Scopes, (1987) Protein Purification: Principles and Practice, SecondEdition (Springer Verlag, N.Y.); and Handbook of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds. 1986).

As used herein, the term “polypeptide” includes any peptide or proteincomprising two or more amino acids joined to each other by peptide bondsor modified peptide bonds, i.e. peptide isosteres. This term refers bothto short chains (peptides and oligopeptides) and to longer chains(proteins).

The polypeptide of the present invention may be in the form of a matureprotein or may be a pre-, pro- or prepro-protein that can be activatedby cleavage of the pre-, pro- or prepro-portion to produce an activemature polypeptide. In such polypeptides, the pre-, pro- orprepro-sequence may be a leader or secretory sequence or may be asequence that is employed for purification of the mature polypeptidesequence.

The polypeptide of the first aspect of the invention may form part of afusion protein. For example, it is often advantageous to include one ormore additional amino acid sequences which may contain secretory orleader sequences, pro-sequences, sequences which aid in purification, orsequences that confer higher protein stability, for example duringrecombinant production. Alternatively or additionally, the maturepolypeptide may be fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol).

Polypeptides may contain amino acids other to the 20 gene-encoded aminoacids, modified either by natural processes, such as bypost-translational processing or by chemical modification techniqueswhich are well known in the art. Among the known modifications which maycommonly be present in polypeptides of the present invention areglycosylation, lipid attachment, sulphation, gamma-carboxylation, forinstance of glutamic acid residues, hydroxylation and ADP-ribosylation.Other potential modifications include acetylation, acylation, amidation,covalent attachment of flavin, covalent attachment of a haeme moiety,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid derivative, covalent attachment ofphosphatidylinositol, cross-lining, cyclization, disulphide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formation, GPI anchorformation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl terminal.In fact, blockage of the amino or carboxyl terminus in a polypeptide, orboth by a covalent modification is common in naturally-occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention.

The modifications that occur in a polypeptide often will be a functionof how the polypeptide is made. For polypeptides that are maderecombinantly, the nature and extent of the modifications in large partwill be determined by the post-translational modification capacity ofthe particular host cell and the modification signals that are presentin the ammo acid sequence of the polypeptide in question. For instance,glycosylation patterns vary between different types of host cell.

The polypeptides of the present invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally-occurringpolypeptides (for example purified from cell culture), recombinantlyproduced polypeptides including fusion proteins), synthetically-producedpolypeptides or polypeptides that are produced by a combination of thesemethods.

The functionally-equivalent polypeptides of the first aspect of theinvention may be polypeptides that are homologous to the INSP085polypeptides. Two poly peptides are said to be “homologous”, as the termis used herein, if the sequence of one of the polypeptides has a highenough degree of identity or similarity to the sequence of the otherpolypeptide. “Identity” indicates that at any particular position in thealigned sequences, the amino acid residue is identical between thesequences. “Similarity” indicates that, at any particular position inthe aligned sequences, the amino acid residue is of a similar typebetween the sequences. Degrees of identity and similarity can be readilycalculated (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing. Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., ad Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991).

Homologous polypeptides therefore include natural biological variants(for example, allelic variants or geographical variations within thespecies from which the polypeptides are derived) and mutants (such asmutants containing amino acid substitutions, insertions or deletions) ofthe INSP085 polypeptides. Such mutants may include polypeptides in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code. Typical such substitutions are among Ala,Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp andGlu; among Asn and Gln; among the basic residues Lys and Arg; or amongthe aromatic residues Phe and Tyr. Particularly preferred are variantsin which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 orjust 1 amino acids are substituted, deleted or added in any combinationEspecially preferred are silent substitutions, additions and deletions,which do mot alter the properties and activities of the protein. Alsoespecially preferred in this regard are conservative substitutions. Suchmutants also include polypeptides in which one or more of the amino acidresidues includes a substituent group.

Typically, greater than 30% identity between two polypeptides isconsidered to be an indication of functional equivalence. Preferably,functionally equivalent polypeptides of the first aspect of theinvention have a degree of sequence identity with the INSP085polypeptide, or with active fragments thereof, of greater than 80%. Morepreferred polypeptides have degrees of identity of greater than 85%,90%, 95%, 98% or 99%, respectively.

The functionally-equivalent polypeptides of the first aspect of theintention may also be polypeptides which have been identified using oneor more techniques of structural alignment. For example, theInpharmatica Genome Threader technology that forms one aspect of thesearch tools used to generate the Biopendium™ search database may beused (see PCT application WO 01/69507) to identify polypeptides ofpresently-unknown function which, while having low sequence identity ascompared to the INSP085 polypeptides, are predicted to be members of theIL-8 like chemokine family, by virtue of sharing significant structuralhomology with the INSP085 polypeptide sequence. By “significantstructural homology” is meant that the Inpharmatica Genome Threaderpredicts two proteins to share structural homology with a certainty of10% and above.

The polypeptides of the first aspect of the invention also includefragments of the INSP085 polypeptides and fragments of the functionalequivalents of the INSP085 polypeptides, provided that those fragmentsare members of the IL-8 like chemokine family or have an antigenicdeterminant in common with the INSP085 polypeptides.

As used herein, the term “fragment” refers to a polypeptide having anamino acid sequence that is the same as part, but not all, of the aminoacid sequence of the INSP085 polypeptide or one of their functionalequivalents. The fragments should comprise at least n consecutive aminoacids from the sequence and, depending on the particular sequence, npreferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 ormere). Small fragments may form an antigenic determinant.

Fragments of the full length INSP085 polypeptides may consist ofcombinations of 2 or 3 of neighbouring exon sequences in the INSP085polypeptide sequences, respectively. For example, such combinationsinclude exons 1 and 2, 2 and 3 or 1, 2 and 3. Such fragments areincluded in the present invention.

Such rents may be “free-standing”, i.e. not part of or fused to otheramino acids or polypeptides, or they may be comprised within a largerpolypeptide of which they form a part or region. When comprised within alarger polypeptide, the fragment of the invention most preferably formsa single continuous region. For instance, certain preferred embodimentsrelate to a fragment having a pre- and/or pro-polypeptide region fusedto the amino terminus of the fragment and/or an additional region fusedto the carboxyl terminus of the fragment. However, several fragments maybe comprised within a single larger polypeptide.

The polypeptides of the present invention or their immunogenic fragments(comprising at least one antigenic determinant) can be used to generateligands, such as polyclonal or monoclonal antibodies, that areimmunospecific for the polypeptides. Such antibodies may be employed toisolate or to identify clones expressing the polypeptides of theinvention or to purity the polypeptides by affinity chromatography. Theantibodies may also be employed as diagnostic or therapeutic aids,amongst other applications, as will be apparent to the skilled reader.

The term “immunospecific” means that the antibodies have substantiallygreater affinity for the polypeptides of the invention than theiraffinity for other related polypeptides in the prior art. As usedherein, the term “antibody” refers to intact molecules as well as tofragments thereof, such as Fab, F(ab)2 and Fv, which are capable ofbinding to the antigenic determinant in question. Such anti-bodies thusbind to the polypeptides of the first aspect of the invention.

By “substantially greater affinity” we mean that there is a measurableincrease in the affinity for a polypeptide of the invention as comparedwith the affinity for known secreted proteins.

Preferably, the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold,100-fold, 10³-fold, 104-fold, 10⁵-fold, 10⁶-fold or greater for apolypeptide of the invention than for known secreted proteins such asmembers of the IL-8 chemokine family of proteins.

If polyclonal antibodies are desired, a selected mammal, such as amouse, rabbit, goat or horse, may be immunised with a polypeptide of thefirst aspect of the invention. The polypeptide used to immunize theanimal cam be derived by recombinant DNA technology or can besynthesized chemically. If desired, the polypeptide can be conjugated toa carrier protein. Commonly used carriers to which the polypeptides maybe chemically coupled include bovine serum albumin, thyrglobulin andkeyhole limpet haemocyanin. The coupled polypeptide is then used toimmunize the animal. Serum from the immunized animal is collected andtreated according to known procedures, for example by immunoaffinitychromatography.

Monoclonal antibodies to the polypeptides of the first aspect of theinvention can also be readily produced by one skilled in the art. Thegeneral methodology for making monoclonal antibodies using hybridomatechnology is well known (see, for example, Kohler, G. and Milstein, C.,Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72(1983); Cole et al., 77-96 in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc. (1985).

Panels of monoclonal antibodies produced against the polypeptides of thefirst aspect of the invention can be screened for various properties,i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies areparticularly useful in purification of the individual polypeptidesagainst which they are directed. Alternatively, genes encoding themonoclonal antibodies of interest may be isolated from hybridomas, forinstance by PCR techniques known in the art, and cloned and expressed inappropriate vectors.

Chimeric antibodies, in which non-human variable regions are joined orfused to human constant regions (see, for example, Liu et al., Proc.Natl. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.

The antibody may be modified to make it less immunogenic in anindividual, for example by humanization (see Jones et al., Nature, 321,522 (1986); Verhoeyen et al., Science, 2390, 1534 (1988); Kabat et al.,J. Immunol., 147, 1709 (1991); Queen et al., Proc. Natl. Acad. Sci. USA,86, 10029 (1989); Gorman et al., Proc. Natl. Acad. Sci-. USA, 88, 34181(1991>; and Hodgson et al., Bio/Technology, 9, 421 (1991)). The term“humanized antibody”, as used herein, refers to antibody molecules inwhich the CDR amino acids and selected other amino acids in the variabledomains of the heavy and/or light chains of a non-human donor antibodyhave been substituted in place of the equivalent amino acids in a humanantibody. The humanized antibody thus closely resembles a human antibodybut has the binding ability of the donor antibody.

In a further alternative, the antibody may be a “bispecific” antibody,that is an antibody having two different antigen binding domains, eachdomain being directed against a different epitope.

Phage display technology may be utilised to select genes which encodeantibodies pith binding activities towards the polypeptides of theinvention either from repertoires of PCR amplified V-genes oflymphocytes from humans screened for possessing the relevant antibodies,or from naive libraries (McCafferty, J. et al., (1990), Nature 348,552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). Theaffinity of these antibodies can also be improved by chain shuffling(Clackson, T. et al., (1991) Nature 352, 624628).

Antibodies generated by the above techniques, whether polyclonal ormonoclonal, have additional utility in that they may be employed asreagents in immunoassays, radioimmunoassays (RIA) or enzyme-linkedimmunosorbent assays (ELISA). In these applications, the antibodies canbe labelled with an analytically-detectable reagent such as aradioisotope, a fluorescent molecule or an enzyme.

Preferred nucleic acid molecules of the second and third aspects of theinvention are those which encode a polypeptide sequence as recited inSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQID NO:12 and functionally equivalent polypeptides. These nucleic acidmolecules may be used in the methods and applications described herein.The nucleic acid molecules of the invention preferably comprise at leastn consecutive nucleotides from the sequences disclosed herein where,depending on the particular sequence, n is 10 or more (for example, 12,14, 15, 18, 20, 25, 30, 35, 40 or more).

The nucleic acid molecules of the invention also include sequences thatare complementary to nucleic acid molecules described above (forexample, for antisense or probing purposes).

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA, or in the form of DNA, including, for instance cDNA,synthetic DNA or genomic DNA. Such nucleic acid molecules may beobtained by cloning, by chemical synthetic techniques or by acombination thereof. The nucleic acid Molecules can be prepared, forexample, by chemical synthesis using techniques such as solid phasephosphoramidite chemical synthesis, from genomic or cDNA libraries or byseparation from an organism. RNA molecules may generally be generated bythe in vitro or in vivo transcription of DNA sequences.

The nucleic acid molecules may be double-stranded or single-stranded.Single-stranded DNA may be the coding strand, also known as the sensestrand, or it may be the non-coding strand, also referred to as theanti-sense strand.

The term “nucleic acid molecule” also includes analogues of DNA and RNA,such as those containing modified backbones, and peptide nucleic acids(PNA). The term “PNA”, as used herein, refers to an antisense moleculeor an anti-gene agent which comprises an oligonucleotide of at leastfive nucleotides in length linked to a peptide backbone of amino acidresidues, which preferably ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in a cell, where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

A nucleic acid molecule which encodes a polypeptide of this inventionmay be identical to the coding sequence of one or more of the nucleicacid molecules disclosed herein.

These molecules also may have a different sequence which, as a result ofthe degeneracy of the genetic code, encodes a polypeptide SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12.Such nucleic acid molecules may include, but are not limited to, thecoding sequence for the mature polypeptide by itself; the codingsequence for the mature polypeptide and additional coding sequences,such as those encoding a leader or secretory sequence, such as a pro-,pre- or prepro-polypeptide sequence; the coding sequence of the maturepolypeptide, with or without the aforementioned additional codingsequences, together with further additional, non-coding sequences,including non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription (includingtermination signals), ribosome binding and mRNA stability. The nucleicacid molecules may also include additional sequences which encodeadditional amino acids, such as those which provide additionalfunctionalities.

The nucleic acid molecules of the second and third aspects of theinvention may also encode the fragments or the functional equivalents ofthe polypeptides and fragments of the first aspect of the invention.Such a nucleic acid molecule may be a naturally-o-occurring variant suchas a naturally-occurring allelic variant, or the molecule may be avariant that is not known to occur naturally. Such non-naturallyoccurring variants of the nucleic acid molecule may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells or organisms.

Among variants in this regard are variants that differ from theaforementioned nucleic acid molecules by nucleotide substitutions,deletions or insertions. The substitutions, deletions or insertions mayinvolve one or more nucleotides. The variants may be altered in codingor non-coding regions or both. Alterations in the coding regions mayproduce conservative or non-conservative amino acid substitutions,deletions or insertions.

The nucleic acid molecules of the invention can also be engineered,using methods generally known in the art, for a variety of reason,including modifying the cloning, processing, and/or expression of thegene product (the polypeptide). DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides areincluded as techniques which may be used to engineer the nucleotidesequences. Site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, introduce mutations and so forth.

Nucleic acid molecules which encode a polypeptide of the first aspect ofthe invention may be ligated to a heterologous sequence so that thecombined nucleic acid molecule encodes a fusion protein. Such combinednucleic acid molecules are included within the second or third aspectsof the invention. For example, to screen peptide libraries forinhibitors of the activity of the polypeptide, it may be useful toexpress, using such a combined nucleic acid molecule, a fusion proteinthat can be recognised by a commercially-available antibody. A fusionprotein may also be engineered to contain a cleavage site locatedbetween the sequence of the polypeptide of the invention and thesequence of a heterologous protein so that the polypeptide may becleaved and purified away from the heterologous protein.

The nucleic acid molecules of the invention also include antisensemolecules that are partially complementary to nucleic acid moleculesencoding polypeptides of the present invention and that thereforehybridize to the encoding nucleic acid molecules (hybridization). Suchantisense molecules, such as oligonucleotides, can be designed torecognise, specifically bind to and prevent transcription of a targetnucleic acid encoding a polypeptide of the inventions as will be knownby those of ordinary skill in the art (see, for example, Cohen, J. S.,Trends in Pharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560(1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et al., Nucleic AcidsRes 6, 3073 (1979); Cooney et at., Science 241, 456 (1988); Dervan etal., Science 251, 1360 (1991).

The term “hybridization” as used here refers to the association of twonucleic acid molecules with one another by hydrogen bonding. Typically,one molecule sill be fixed to a solid support and the other will be freein solution. Then, the two molecules may be placed in contact with oneanother under conditions that favour hydrogen bonding. Factors thataffect this bonding include: the type and volume of solvent; reactiontemperature; time of hybridization; agitation; agents to block thenon-specific attachment of the liquid phase molecule to the solidsupport (Denhardt's reagent or BLOTTO); the concentration of themolecules; use of compounds to increase the rate of association ofmolecules (dextran sulphate or polyethylene glycol); and the stringencyof the washing conditions following hybridization (see Sambrook et al.[supra]).

The inhibition of hybridization of a completely complementary moleculeto a target molecule may be examined using a hybridization assay, asknown in the art (see, for example, Sambrook et al. [supra]). Asubstantially homologous molecule will then compete for and inhibit thebinding of a completely homologous molecule to the target molecule undervarious conditions of stringency, as taught in Wahl, G. M. and S. L.Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R (1987;Methods Enzymol. 152:507-511).

“Stringency” refers to conditions in a hybridization reaction thatfavour the association of very similar molecules over association ofmolecules that differ. High stringency hybridisation conditions aredefined as overnight incubation at 42° C. in a solution comprising 50%foramide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH7.6), 5×Denhardts solution, 10% dextran sulphate, and 20microgram/ml denatured, sheared salmon sperm DNA, followed by washingthe filters in 0.1×SSC at approximately 65° C. Low stringency conditionsinvolve the hybridisation reaction being carried out at 35° C. (seeSambrook et al. [supra]). Preferably, the conditions used forhybridization are those of high stringency.

Preferred embodiments of this aspect of the invention are nucleic acidmolecules that are at least 70% identical over their entire length to anucleic acid molecule encoding the INSP085 polypeptides and nucleic acidmolecules that are substantially complementary to such nucleic acidmolecules. Preferably, a nucleic acid molecule according to this aspectof the invention comprises a region that is at least 80% identical overits entire length to such coding sequences, or is a nucleic acidmolecule that is complementary thereto. In this regard, nucleic acidmolecules at least 90%, preferably at least 95%, more preferably atleast 98%, 99% or more identical over their entire length to the sameare particularly preferred. Preferred embodiments in this respect arenucleic acid molecules that encode polypeptides which retainsubstantially the same biological function or activity as the INSP085polypeptides.

The invention also provides a process for detecting a nucleic acidmolecule of the invention, comprising the steps of: (a) contacting anucleic probe according to the invention with a biological sample underhybridizing conditions to form duplexes; and (b) detecting any suchduplexes that are formed.

As discussed additionally below in connection with assays that may beutilised according to the invention, a nucleic acid molecule asdescribed above may be used as a hybridization probe for RNA, cDNA orgenomic DNA, in order to isolate full-length cDNAs and genomic clonesencoding the INSP085 polypeptides and to isolate cDNA and genomic clonesof homologous or orthologous genes that have a high sequence similarityto the gene encoding this polypeptide.

In this regard, the following techniques, among others known in the art,may be utilised and are discussed below for purposes of illustration.Methods fox DNA sequencing and analysis are well known and are generallyavailable in the art and may, indeed, be used to practice many of theembodiments of the invention discussed herein. Such methods may employsuch enzymes as the Klenow fragment of DNA polymerase I, Sequenase (USBiochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin Elmer),thermostable T7 polymerase (Amersham, Chicago, Ill.), or combinations ofpolymerases and proof-reading exonucleases such as those found in theELONGASE Amplification System marketed by Gibco/BRL (Gaithersburg, Md.).Preferably, the sequencing process may be automated using machines suchas the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), the PeltierThermal Cycler PTC200; MJ Research, Watertown, Mass.) and the ABICatalyst and 373 and 377 DNA Sequencers (Perkin Elmer).

One method for isolating a nucleic acid molecule encoding a polypeptidewith an equivalent function to that of the INSP085 polypeptide is toprobe a genomic or cDNA library with a natural or artificially-designedprobe using standard procedures that are recognised in the art (see, forexample, “Current Protocols in Molecular Biology”, Ausubel et al. (eds).Greene Publishing Association and John Wiley Interscience, New York,1989, 1992). Probes comprising at least 15, preferably at least 30, andmore preferably at least 50, contiguous bases that correspond to, or arecomplementary to, nucleic acid sequences from the appropriate encodinggene (SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9and SEQ ID NO:11), are particularly useful probe. Such probes may belabelled with an analytically-detectable reagent to facilitate theiridentification. Useful reagents include, but are not limited to,radioisotopes, fluorescent dyes and enzymes that are capable ofcatalysing the formation of a detectable product. Using these probes,the ordinarily skilled artisan will be capable of isolatingcomplementary copies of genomic DNA, cDNA or RNA polynucleotidesencoding proteins of interest from human, mammalian or other animalsources and screening such sources for related sequences, for example,for additional members of the family, type and/or subtype.

In many cases, isolated cDNA sequences will be incomplete, in that theregion encoding the polypeptide Mill be cut short, normally at the 5′end. Several methods are available to obtain full length cDNAs, or toextend short cDNAs. Such sequences may be extended utilising a partialnucleotide sequence and employing various methods known in the art todetect upstream sequences such as promoters and regulatory elements. Forexample, one method which may be employed is based on the method ofRapid Amplification of cDNA Ends (RACE; see, for example, Frohman etal., PNAS USA 85, 8998-9002, 1988) Recent modifications of thistechnique, exemplified by the Marathon™ technology (ClontechLaboratories Inc.), for example, have significantly simplified thesearch for longer cDNAs. A slightly different technique, termed“restriction-site” PCR, uses universal primers to retrieve unknownnucleic acid sequence adjacent a known locus (Sarkar, G. (1993) PCRMethods Applic. 2:318-322). Inverse PCR may also be used to amplify orto extend sequences using divergent primers based on a known region(Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). Another methodwhich may be used is capture PCR which involves PCR amplification of DNAfragments adjacent a known sequence in human and yeast artificialchromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic., 1,111-119). Another method which may be used to retrieve unknown sequencesis that of Parker, J. D. et al. (1991); Nucleic Acids Res.19:3055-3060). Additionally, one may use PCR, nested primers, andPromoterFinder™ libraries to walk genomic DNA (Clontech, Palo Alto,Calif.) This process avoids the need to screen libraries and is usefulin finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use Librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences that contain the 5′ regions of genes. Use of a randomly primedlibrary may be especially preferable for situations in which an oligod(T) library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

In one embodiment of the invention, the nucleic acid molecules of thepresent invention may be used for chromosome localisation. In thistechnique, a nucleic acid molecule is specifically targeted to, and canhybridize with, a particular location on an individual human chromosome.The mapping of relevant sequences to chromosomes according to thepresent invention is an important step in the confirmatory correlationof those sequences with the gene-associated disease. Once a sequence hasbeen mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be correlated with genetic map data.Such data are found in, for example, V. McKusick, Mendelian Inheritancein Man (available on-line through Johns Hopkins University Welch MedicalLibrary). The relationships between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes). Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localised by genetic linkage toa particular genomic region, any sequences mapping to that area mayrepresent associated or regulatory genes for finer investigation. Thenucleic acid molecule may also be used to detect differences in thechromosomal location due to translocation, inversion, etc. among normal,carrier, or affected individuals.

The nucleic acid molecules of the present invention are also valuablefor tissue localisation. Such techniques allow the determination ofexpression patterns of the polypeptide in tissues by detection of themRNAs that encode them. These techniques include in situ hybridizationtechniques and nucleotide amplification techniques, such as PCR. Resultsfrom these studies provide an indication of the normal functions of thepolypeptide in the organism. In addition, comparative studies of thenormal expression pattern of mRNAs with that of mRNAs encoded by amutant gene provide valuable insights into the role of mutantpolypeptides in disease. Suck inappropriate expression may be of atemporal, spatial or quantitative nature.

Gene silencing approaches may also be undertaken to down-regulateendogenous expression of a gene encoding a polypeptide of the invention.RNA interference (RNAi) (Elbashir, S M et al., Nature 2001, 411,494-498) is one Method of sequence specific post-transcriptional genesilencing that may be employed. Short dsRNA oligonucleotides aresynthesized in vitro and introduced into a cell. The sequence specificbinding of these dsRNA oligonucleotides triggers the degradation oftarget mRNA, reducing or ablating target protein expression.

Efficacy of the gene silencing approaches assessed above may be assessedthrough the measurement of polypeptide expression (for example, byWestern blotting), and at the RNA level using TaqMan-basedmethodologies.

The vectors of the present invention comprise nucleic acid molecules ofthe invention and may be cloning or expression vectors. The host cellsof the invention, which may be transformed, transfected or transducedwith the vectors of the invention may be prokaryotic or eukaryotic.

The polypeptides of the invention may be prepared in recombinant form byexpression of their encoding nucleic acid molecules in vectors containedwithin a host cell. Such expression methods are well known to those ofskill in the art and many are described in detail by Sambrook et al.(supra) and Fernandez & Hoeffler (1998, eds. “Gene expression systems.Using nature for the art of expression”. Academic Press, San Diego,London, Boston, New York, Sydney, Tokyo, Toronto).

Generally, any system or vector that is suitable to maintain, propagateor express nucleic acid molecules to produce a polypeptide in therequired host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those described in Sambrook etal., (supra). Generally, the encoding gene can be placed under thecontrol of a control element such as a promoter, ribosome binding site(for bacterial expression) and, optionally, an operator, so that the DNAsequence encoding the desired polypeptide is transcribed into RNA in thetransformed host cell.

Examples of suitable expression systems include, for example,chromosomal, episomal and virus-derived systems, including, for example,vectors derived from: bacterial plasmids, bacteriophage, transposons,yeast episomes, insertion elements, yeast chromosomal elements, virusessuch as baculoviruses, papova viruses such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,or combinations thereof, such as those derived from plasmid andbacteriophage genetic elements, including cosmids and phagemids. Humanartificial chromosome s (HACs) may also be employed to deliver largerfragments of DNA than can be contained and expressed in a plasmid.

Particularly suitable expression systems include microorganisms such asbacteria transformed with recombinant bacteriophage, plasmid or cosmidDNA expression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (for example,baculovirus); plant cell systems transformed with virus expressionvectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or with bacterial expression vectors (for example, Ti orpBR322 plasmids); or animal cell systems. Cell-free translation systemscan also be employed to produce the polypeptides of the invention.

Introduction of nucleic acid molecules encoding a polypeptide of thepresent invention into host cells can be effected by methods describedin many standard laboratory manuals, such as Davis et al., Basic Methodsin Molecular Biology (1986) and Sambrook et al., (supra). Particularlysuitable methods include calcium phosphate transfection, DEAE-dextranmediated transfection, transfection, microinjection, cationiclipid-mediated transfection, electroporation, transduction, scrapeloading, ballistic introduction or infection (see Sambrook et al, 1989[supra]; Ausubel et al., 1991 [supra]; Spector, Goldman & Leinwald,1998). In eukaryotic cells, expression systems may either be transient(for example, episomal) or permanent (chromosomal integration) accordingto the needs of the system.

The encoding nucleic acid molecule may or may not include a sequenceencoding a control sequence, such as a signal peptide or leadersequence, as desired, for example, for secretion of the translatedpolypeptide into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment. These signalsmay be endogenous to the polypeptide or they may be heterologoussignals. Leader sequences can be removed by the bacterial host inpost-translational processing.

In addition to control sequences, it may be desirable to add regulatorysequences that allow for regulation of the expression of the polypeptiderelative to the growth of the host cell. Examples of regulatorysequences are those which use the expression of a gene to be increasedor decreased in response to a chemical or physical stimulus, includingthe presence of a regulatory compound or to various temperature ormetabolic conditions. Regulatory sequences are those non-translatedregions of the vector, such as enhancers, promoters and 5′ and 3′untranslated regions. These interact with host cellular proteins tocarry out transcription and translation. Such regulatory sequences mayvary in their strength and specificity. Depending on the vector systemand host utilised, any number of suitable transcription and translationelements, including constitutive and inducible promoters, may be used.For example, when cloning in bacterial systems, inducible promoters suchas the hybrid lacZ promoter of the Bluescript phagemid (Stratagene,LaJolla, Calif.) or pSport™ plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (forexample, heat shock, RUBISCO and storage protein genes) or from plantviruses (for example, viral promoters or leader sequences) may be clonedinto the vector. In mammalian cell systems, promoters from mammaliangenes or from mammalian viruses are preferable. If it is necessary togenerate a cell line that contains multiple copies of the sequence,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

An expression vector is constructed so that the particular nucleic acidcoding sequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the regulatory sequences being such that the coding sequenceis transcribed under the “control” of the regulatory sequences, i.e.,RNA polymerase which binds to the DNA molecule at the control sequencestranscribes the coding sequence. In some cases it may be necessary tomodify the sequence so that it may be attached to the control sequenceswith the appropriate orientation; i.e., to maintain the reading frame.

The control sequences and other regulatory sequences may be ligated tothe nucleic acid coding sequence prior to insertion into a vector.Alternatively, the coding sequence can be cloned directly into anexpression vector that already contains the control sequences and anappropriate restriction site.

For long-term, high-yield production of a recombinant polypeptide,stable expression is preferred. For example, cell lines which stablyexpress the polypeptide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells that successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalised cell lines available from the AmericanType Culture Collection (ATCC) including, but not limited to, Chinesehamster ovary (CHO)), HeLa, baby hamster kidney (BWH, monkey kidney(COS), C127, 3T3, BHK, HEK293, Bowes melanoma and human hepatocellularcarcinoma (for example Hep G2) cells and a number of other cell lines.

In the baculovirus system, the materials for baculovirus/insect cellexpression systems are commercially available in kit form, inter alia,Invitrogen, San Diego Calif. (the “MaxBac” kit). These techniques aregenerally known to those skilled in the art and are described fully inSummers and Smith Texas Agricultural Experiment Station Bulletin No.1555 (1987). Particularly suitable host cells for use in this systeminclude insect cells such as Drosophila 82 and Spodoptera Sf9 cells.

There are many plant cell culture and whole plant genetic expressionsystems known in the art. Examples of suitable plant cellular geneticexpression systems include those described in U.S. Pat. No. 5,693,506;U.S. Pat. No. 5,659,122; and U.S. Pat. No. 5,608,143. Additionalexamples of genetic expression in plant cell culture has been describedby Zenk, Phytochemistry 30, 3861-3863 (1991).

In particular, all plants from which protoplasts can be isolated andcultured to give whole regenerated plants can be utilised, so that wholeplants are recovered which contain the transferred gene. Practically allplants can be regenerated from cultured cells or tissues, including butnot limited to all major species of sugar cane, sugar beet, cotton,fruit and other trees, legumes and vegetables.

Examples of particularly preferred bacterial host cells includestreptococci staphylococci, E. coli, Streptomyces and Bacillus subtiliscells.

Examples of particularly suitable host cells for fungal expressioninclude yeast cells (for example, S. cerevisiae) and Aspergillus cells.

Any number of selection systems are known in the art that may be used torecover transformed cell lines. Examples include the herpes simplervirus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) andadenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell22:817-23) genes that can be employed in tk⁻ or apr^(±) cells,respectively.

Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dihydrofolate reductase (DHFR)that confers resistance to methotrexate (Wigler, M. et al (1980) Proc.Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to theaminoglycosides neomycin and G418 (Colbere-(Garapin, F. et al. (1981) J.Mol. Diol. 150:1-14) and also or pat, which confer resistance tochlorsulfuron and phosphinothricin acetyltransferase, respectively.Additional selectable genes have bean described, examples of which willbe clear to those of skill in the art.

Although the presence or absence of marker gene expression suggests thatthe gene C of interest is also present, its presence and expression mayneed to be confirmed. For example, if the relevant sequence is insertedwithin a marker gene sequence, transformed cells containing theappropriate sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding a polypeptide of the invention under the control of asingle promoter. Expression of be marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

Alternatively, host cells that contain a nucleic acid sequence encodinga polypeptide of the invention and which express said polypeptide may beidentified by a variety of procedures known to those of skill in theart. These procedures include, but are not limited to, DNA-DNA orDNA-RNA hybridizations and protein bioassays, for example, fluorescenceactivated cell sorting (FACS) or immunoassay techniques (such as theenzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]),that include membrane, solution, or chip based technologies for thedetection and/or quantification of nucleic acid or protein (see Hampton,R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D Y et al. (1983) J. Exp. Med. 158, 1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labelled hybridization or PCR probesfor detecting sequences related to nucleic acid molecules encodingpolypeptides of the present invention include oligolabelling, nicktranslation, end-labelling or PCR amplification using a labelledpolynucleotide. Alternatively, the sequences encoding the polypeptide ofthe invention may be cloned into a vector for the production of an mRNAprobe. Such vectors are known in the art, are commercially available,and may be used to synthesise RNA probes in vitro by addition of anappropriate RNA polymerase such as T7, T3 or SP6 and labellednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland, Ohio)).

Suitable reporter molecules or labels, which may be used for ease ofdetection, include radionuclides, enzymes and fluorescent,chemiluminescent or chromogenic agents as well as substrates, cofactors,inhibitors, magnetic particles, and the like.

Nucleic acid molecules according to the present invention may also beused to create transgenic animals, particularly rodent animals. Suchtransgenic animals form a further aspect of the present invention. Thismay be done locally by modification of somatic cells, or by germ linetherapy to incorporate heritable modifications. Such transgenic animalsmay be particularly useful in the generation of animal models for drugmolecules effective as modulators of the polypeptides of the presentinvention.

The polypeptide can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulphate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromotography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography is particularlyuseful for purification. Well known techniques for refolding proteinsmay be employed to regenerate an active conformation when thepolypeptide is denatured during isolation and or purification.

Specialised vector constructions may also be used to facilitatepurification of protein, as desired, by joining sequences encoding thepolypeptides of the invention to a nucleotide sequence encoding apolypeptide domain that will facilitate purification of solubleproteins. Examples of such purification-facilitating domains includemetal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilised metals, protein A domains that allowpurification on immobilised immunoglobulin, and the domain utilised inthe FLAGS extension/affinity purification system (Immunex Corp.,Seattle, Wash.). The inclusion of cleavable linker sequences such asthose specific for Factor XA or enterokinase (Invitrogen, San Diego,Calif.) between the purification domain and the polypeptide of theinvention may be used to facilitate purification. One such expressionvector provides for expression of a fusion protein containing thepolypeptide of the invention fused to several histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification by IMAC (immobilised metal ion affinitychromatography as described in Porath, J. et al (1992), Prot. Exp.Purif. 3: 263-281) while the thioredoxin or enterokinase cleavage siteprovides a mean-s for purifying the polypeptide from the fusion protein.A discussion of vectors which contain fusion proteins is provided inKroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

If the polypeptide is to be expressed for use in screening assays,generally it is preferred that it be produced at the surface of the hostcell in which it is expressed. In this event, the host cells may beharvested prior to use in the screening assay, for example usingtechniques such as fluorescence activated cell sorting (FACS) orimmunoaffinity techniques. If the polypeptide is secreted into themedium, the medium can be recovered in order to recover and purify theexpressed polypeptide. If polypeptide is produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

The polypeptide of the invention cam be used to screen libraries ofcompounds in any of a variety of drug screening techniques. Suchcompounds may activate (agonise) or inhibit (antagonize) the level ofexpression of the gene or the activity of the polypeptide of theinvention and form a further aspect of the present invention Preferredcompounds are effective to alter the expression of a natural gene whichencodes a polypeptide of the first aspect of the invention or toregulate the activity of a polypeptide of the first aspect of theinvention.

Agonist or antagonist compounds may be isolated from, for example,cells, cell-free preparations, chemical libraries or natural productmixtures. These agonists or antagonists may be natural or modifiedsubstrates, ligands, enzymes, receptors or structural or functionalmimetics. For a suitable review of such screening techniques, seeColigan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991).

Compounds that are most likely to be good antagonists are molecules thatbind to the polypeptide of the invention without inducing the biologicaleffects of the polypeptide upon binding to it. Potential antagonistsinclude small organic molecules, peptides, polypeptides and antibodiesthat bind to the polypeptide of the invention and thereby inhibit orextinguish its activity. In this fashion, binding of the polypeptide tonormal cellular binding molecules may be inhibited, such that the normalbiological activity of the polypeptide is prevented.

The polypeptide of the invention that is employed in such a screeningtechnique may be free in solution, affixed to a solid support, borne ona cell surface or located intracellularly. In general, such screeningprocedures may involve using appropriate cells or cell membranes thatexpress the polypeptide that are contacted with a test compound toobserve binding, or stimulation or inhibition of a functional response.The functional response of the cells contacted with the test compound isthen compared with control cells that were not contacted with the testcompound. Such an assay may assess whether the test compound results ina signal generated by activation of the polypeptide, using anappropriate detection system. Inhibitors of activation are generallyassayed in the presence of a known agonist and the effect on activationby the agonist in the presence of the test compound is observed.

A preferred method for identifying an agonist or antagonist compound ofa poly peptide of the present invention comprises:

(a) contacting a cell expressing on the surface thereof the polypeptideaccording to the first aspect of the invention, the polypeptide beingassociated with a second component capable of providing a detectablesignal in response to the binding of a compound to the polypeptide, witha compound to be screened under conditions to permit binding to thepolypeptide; and(b) determining whether the compound binds to and activates or inhibitsthe polypeptide by measuring the level of a signal generated from theinteraction of the compound with the polypeptide.

A further preferred method for identifying an agonist or antagonist of apolypeptide of the invention comprises:

(a) contacting a cell expressing on the surface thereof the polypeptide,the polypeptide being associated with a second component capable ofproviding a detectable signal in response to the binding of a compoundto the polypeptide, with a compound to be screened under conditions topermit binding to the polypeptide; and(b) determining whether the compound binds to and activates or inhibitsthe polypeptide by comparing the level of a signal generated from theinteraction of the compound with the polypeptide with the level of asignal in the absence of the compound.

In further preferred embodiments, the general methods that are describedabove may further comprise conducting the identification of agonist orantagonist in the presence of labelled or unlabelled ligand for thepolypeptide.

In another embodiment of the method for identifying an agonist orantagonist of a polypeptide of the present invention comprises:

determining the inhibition of binding of a ligand to cells which have apolypeptide of the invention on the surface thereof, or to cellmembranes containing such a polypeptide, in the presence of a candidatecompound under conditions to permit Winding to the polypeptide, anddetermining the amount of ligand bound to the polypeptide. A compoundcapable of causing reduction of binding of a ligand is considered to bean agonist or antagonist. Preferably the ligand is labelled.

More particularly, a method of screening for a polypeptide antagonist oragonist compound comprises the steps of:

(a) incubating a labelled ligand with a whole cell expressing apolypeptide according to the invention on the cell surface, or a cellmembrane containing a polypeptide of the invention,(b) measuring the amount of labelled ligand bound to the whole cell orthe cell membrane;(c) adding a candidate compound to a mixture of labelled ligand and thewhole cell or the cell membrane of step (a) and allowing the mixture toattain equilibrium;(d) measuring the amount of labelled ligand bound to the whole cell orthe cell membrane after step (c); and(e) comparing the difference in the labelled ligand bound in step (b)and (d), such that the compound which causes the reduction in binding instep (d) is considered to be an agonist or antagonist.

The INSP085 polypeptides may also be found to modulate immune and/ornervous system cell proliferation and differentiation in adose-dependent manner in the above-described assays. Thus, the“functional equivalents” of the INSP085 polypeptides includepolypeptides that exhibit any of the same growth and differentiationregulating activities in the above-described assays in a dose-dependentmanner. Although the degree of dose-dependent activity need not beidentical to that of the INSP085 polypeptides, preferably the“functional equivalents” will exhibit substantially similardose-dependence in a given activity assay compared to the INSP085polypeptides.

In certain of the embodiments described above, simple binding assays maybe used, in which the adherence of a test compound to a si face bearingthe polypeptide is detected by means of a label directly or indirectlyassociated with the test compound or in an assay involving competitionwith a labelled competitor. In another embodiment, competitive drugscreening assays may be used, in which neutralizing antibodies that arecapable of binding the polypeptide specifically compete with a testcompound for binding. In this manner, the antibodies can be used todetect the presence of any test compound that possesses specific bindingaffinity for the polypeptide.

Assays may also be designed to detect the effect of added test compoundson the production of mRNA encoding the polypeptide in cells. Forexample, an ELISA may be constructed that measures secreted orcell-associated levels of polypeptide using monoclonal or polyclonalantibodies by standard methods known in the art, and this can be used tosearch for compounds that may inhibit or enhance the production of thepolypeptide from suitably manipulated cells or tissues. The formation ofbinding complexes between the polypeptide and the compound being testedmay then be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe polypeptide of interest (see International patent applicationWO84/03564). In this method, large numbers of different small testcompounds are synthesised on a solid substrate, which may then bereacted with the polypeptide of the invention and washed. One way ofimmobilising the polypeptide is to use non-neutralizing antibodies.Bound polypeptide may then be detected using methods that are well knownin the art. Purified polypeptide can also be coated directly onto platesfor use in the aforementioned drug screening techniques.

The polypeptide of the invention may be used to identify membrane-boundor soluble receptors, through standard receptor binding techniques thatare known in the art, such as ligand binding and crosslinking assays inwhich the polypeptide is labelled with a radioactive isotope, ischemically modified, or is fused to a peptide sequence that facilitatesits detection or purification, and incubated with a source of theputative receptor (for example, a composition of cells, cell membranes,cell supernatants, tissue extracts, or bodily fluids). The efficacy ofbinding may be measured using biophysical techniques such as surfaceplasmon resonance and spectroscopy. Binding assays may be used for thepurification and cloning of the receptor, bat may also identify agonistsand antagonists of the polypeptide, that compete with the binding of thepolypeptide to its receptor. Standard methods for conducting screeningassays are well understood in the art.

The invention also includes a screening kit useful in the methods foxidentifying agonists, antagonists, ligands, receptors, substrates,enzymes, that are described above.

The invention includes the agonists, antagonists, ligands, receptors,substrates and enzymes, and other compounds which modulate the activityor antigenicity of the polypeptide of the invention discovered by themethods that are described above.

The invention also provides pharmaceutical compositions comprising apolypeptide, nucleic acid, ligand or compound of the invention incombination with a suitable pharmaceutical carrier. These compositionsmay be suitable as therapeutic or diagnostic reagents, as vaccines, oras other immunogenic compositions, as outlined in detail below.

According to the terminology used herein, a composition containing apolypeptide, nucleic acid, ligand or compound [X] is “substantially freeof” impurities [herein, Y] when at least 85% by weight of the total X+Yin the composition is X. Preferably, X comprises at least about 90% byweight of the total of X+Y in the composition, more preferably at leastabout 95%, 98% or even 99% by weight.

The pharmaceutical compositions should preferably comprise atherapeutically effective amount of the polypeptide, nucleic acidmolecule, ligand, or compound of the invention. The term“therapeutically effective amount” as used herein refers to an amount ofa therapeutic agent needed to treat, ameliorate, or prevent a targeteddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. For any compound, the therapeutically effectivedose can be estimated initially either in cell culture assays, forexample, of neoplastic cells, or in animal models, usually mice,rabbits, dogs, or pigs. The animal model may also be used to determinethe appropriate concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans.

The precise effective amount for a human subject will depend upon theseverity of the disease state, general health of the subject, age,weight, and gender of the subject, diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. This amount can be determined by routineexperimentation and is within the judgement of the clinician. Generally,an effective dose will be from 0_(—)01 mg/kg to 50 mg/kg, preferably0.05 mg/kg to 10 mg/kg Compositions may be administered individually toa patient or may be administered in combination with other agents, drugsor hormones.

A pharmaceutical composition may also contain a pharmaceuticallyacceptable carrier, for administration of a therapeutic agent. Suchcarriers include antibodies and other polypeptides, genes and othertherapeutic agents such as liposomes, provided that the carrier does notitself induce the production of antibodies harmful to the individualreceiving the composition, and which may be administered without unduetoxicity. Suitable carriers may be large, slowly metabolisedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers andinactive virus particles.

Pharmaceutically acceptable salts can be used therein, for example,mineral acid salts such as hydrochlorides, hydrobromaides, phosphates,sulphates, and be like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. A thorough discussionof pharmaceutically acceptable carriers is available in Remington'sPharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, may bepresent in such compositions. Such carriers enable the pharmaceuticalcompositions to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, foringestion by the patient.

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals; inparticular, human subjects can be treated.

The pharmaceutical compositions utilised in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal or transcutaneousapplications (for example, see WO98/20734), subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, intravaginalor rectal means. Gene guns or hyposprays may also be used to administerthe pharmaceutical compositions of the invention. Typically, thetherapeutic compositions may be prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection may also beprepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Dosage treatmentmay be a single dose schedule or a multiple dose schedule.

If the activity of the polypeptide of the invention is in excess in aparticular disease state, several approaches are available. One approachcomprises administering to a subject an inhibitor compound (antagonist)as described above, along with a pharmaceutically acceptable carrier inan amount effective to inhibit the function of the polypeptide, such asby blocking the binding of ligands, substrates, enzymes, receptors, orby inhibiting a second signal, and thereby alleviating the abnormalcondition. Preferably, such antagonists are antibodies. Most preferably,such antibodies are chimeric and/or humanized to minimise theirimmunogenicity, as described previously.

In another approach, soluble forms of the polypeptide that retainbinding affinity for the ligand, substrate, enzyme, receptor, inquestion, may be administered. Typically, the polypeptide may beadministered in the form of fragments that retain the relevant portions.

In an alternative approach, expression of the gene encoding thepolypeptide can be inhibited using expression blocking techniques, suchas the use of antisense nucleic acid molecules (as described above),either internally generated or separately administered Modifications ofgene expression can be obtained by designing complementary sequences orantisense molecules (DNA, RNA, or PNA) to the control, 5′ or regulatoryregions (signal sequence, promoters, enhancers and introns) of the geneencoding the polypeptide. Similarly, inhibition can be achieved using“triple helix” base-pairing methodology Triple helix pairing is usefulbecause it causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt Kisco, N.Y.). The complementary sequence on antisensemolecule may also be designed to block translation of mRNA by preventingthe transcript from binding to ribosomes. Such oligonucleotides may beadministered or may be generated in situ from expression in vivo.

In addition, expression of the polypeptide of the invention may beprevented by using ribozymes specific to its encoding mRNA sequence.Ribozymes are catalytically active RNAs that can be natural or synthetic(see for example Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4),527-33). Synthetic ribozymes can be designed to specifically cleavemRNAs at selected positions thereby preventing translation of the mRNAsinto functional polypeptide. Ribozymes may be synthesised with a naturalribose phosphate backbone and natural bases, as normally found in RNAmolecules. Alternatively the ribozymes may be synthesised withnon-natural backbones, for example, 2′-O-methyl RNA, to provideprotection from ribonuclease degradation and may contain modified bases.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the moleculeor the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of non-traditional bases such asinosine, queosine and butosine, as well as acetyl-, methyl-, thio- andsimilarly modified forms of adenine, cytidine, guanine, thymine anduridine which are not as easily recognized by endogenous endonucleases.

For treating abnormal conditions related to an under-expression of thepolypeptide of the invention and its activity, several approaches arealso available. One approach comprises administering to a subject atherapeutically effective amount of a compound that activates thepolypeptide, i.e., an agonist as described above, to alleviate theabnormal condition. Alternatively, a therapeutic amount of thepolypeptide in combination with a suitable pharmaceutical carrier may beadministered to restore the relevant physiological balance ofpolypeptide.

Gene therapy may be employed to effect the endogenous production of thepolypeptide by the relevant cells in the subject Gene therapy is used totreat permanently the inappropriate production of the polypeptide byreplacing a defective gene with a corrected therapeutic gene.

Gene therapy of the present invention can occur in vivo or ex vivo. Exvivo gene therapy requires the isolation and purification of patientcells, the introduction of a therapeutic gene and introduction of thegenetically altered cells back into the patient. In contrast, in vivogene therapy does not require isolation and purification of a patientscells.

The therapeutic gene is typically “packaged” for administration to apatient. Gene delivery vehicles may be non-viral, such as liposomes, orreplication-deficient viruses, such as adenovirus as described byBerkner, K. L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) oradeno-associated virus (AAV) vectors as described by Muzyczka, N., inCurr. Top. Microbiol. Immunol., 158, 97-129 (1992) and U.S. Pat. No.5,252,479. For example, a nucleic acid Molecule encoding a polypeptideof the invention may be engineered for expression in areplication-defective retroviral vector. This expression construct maythen be isolated and introduced into a packaging cell transduced with aretroviral plasmid vector containing RNA encoding the polypeptide, suchthat the packaging cell now produces infectious viral particlescontaining the gene of interest These producer cells may be administeredto a subject for engineering cells in vivo and expression of thepolypeptide in vivo (see Chapter 20, Gene Therapy and other MolecularGenetic-based Therapeutic Approaches, (and references cited therein) inHuman Molecular Genetics (1996), T Stracham and A P Read, BIOSScientific Publishers Ltd).

Another approach is the administration of “naked DNA” in which thetherapeutic gene is directly injected into the bloodstream or muscletissue.

In situations in which the polypeptides or nucleic acid molecules of theinvention are disease-causing agents, the invention provides that theycan be used in vaccines to raise antibodies against the disease causingagent.

Vaccines according to the invention may either be prophylactic (i.e. toprevent infection) or therapeutic (i.e. to treat disease afterinfection). Such vaccines comprise immunizing antigen(s), immunogen(s),polypeptide(s), protein(s) or nucleic acid, usually in combination withpharmaceutically-acceptable carriers as described above, which includeany carrier that does not itself induce the production of antibodiesharmful to the individual receiving the composition. Additionally, thesecarriers may function as immunostimulating agents (“adjuvants”).Furthermore, the antigen or immunogen may be conjugated to a bacterialtoxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori,and other pathogens.

Since polypeptides may be broken down in the stomach, vaccinescomprising polypeptides are preferably administered parenterally (forinstance, subcutaneous, intramuscular, intravenous, or intradermalinjection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the recipient, and aqueous andnor-aqueous sterile suspensions which may include suspending agents orthickening agents.

The vaccine formulations of the invention may be presented in unit-doseor multi-dose containers. For example, sealed ampoules and vials and maybe stored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

Genetic delivery of antibodies that bind to polypeptides according tothe invention may also be effected, for example, as described inInternational patent application WO98/55607.

The technology referred to as jet injection (see, for example,www.powderject.com) may also be useful in the formulation of vaccinecompositions.

A number of suitable methods for vaccination and vaccine deliverysystems are described in International patent application WO00/29428.

This invention also relates to the use of nucleic acid moleculesaccording to the present invention as diagnostic reagents. Detection ofa mutated form of the gene characterised by the nucleic acid moleculesof the invention which is associated with a dysfunction will provide adiagnostic tool that can add to, or define, a diagnosis of a disease, orsusceptibility to a disease, which results from under-expression,over-expression or altered spatial or temporal expression of the gene.Individuals carrying mutations in the gene may be detected at the DNAlevel by a variety of techniques.

Nucleic acid molecules for diagnosis may be obtained from a subjectssells, such as from blood, urine, saliva, tissue biopsy or autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR, ligase chain reaction (LCR), standdisplacement amplification (SDA), or other amplification techniques (seeSaiki et al., Nature, 324, 163-166 (1986); Bej, et al., Crit. Rev.Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al., J. Virol.Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294(1990)) prior to analysis.

In one embodiment, this aspect of the invention provides a method ofdiagnosing a disease in a patient, comprising assessing the level ofexpression of a natural gene encoding a polypeptide according to theinvention and comparing said level of expression to a control level,wherein a level that is different to said control level is indicative ofdisease. The method may comprise the steps of:

a) contacting a sample of tissue from the patient with a nucleic acidprobe under stringent conditions that allow the formation of a hybridcomplex between a nucleic acid molecule of the invention and the probe;b) contacting a control sample with said probe under the same conditionsused in step a);c) and detecting the presence of hybrid complexes in said samples;wherein detection of levels of the hybrid complex in the patient samplethat differ from levels of the hybrid complex in the control sample isindicative of disease.

A further aspect of the invention comprises a diagnostic methodcomprising the steps of:

a) obtaining a tissue sample from a patient being tested for disease,b) isolating a nucleic acid molecule according to the invention fromsaid tissue sample; andc) diagnosing the patient for disease by detecting the presence of amutation in the nucleic acid molecule which is associated with disease.

To aid the detection of nucleic acid molecules in the above-describedmethods, an amplification step, for example using PCR, may be included.

Deletions and insertions can be detected by a change in the size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to labelled RNA of theinvention or alternatively, labelled antisense DNA sequences of theinvention. Perfectly-matched sequences can be distinguished frommismatched duplexes by RNase digestion or by assessing differences inmelting temperatures. The presence or absence of the mutation in thepatient may be detected by contacting DNA with a nucleic acid probe thathybridises to the DNA under stringent conditions to form a hybriddouble-stranded molecule, the hybrid double-stranded molecule having anunhybridized portion of the nucleic acid probe strand at any portioncorresponding to a mutation associated with disease; and detecting thepresence or absence of an unhybridized portion of the probe strand as anindication of the presence or absence of a disease-associated mutationin the corresponding portion of the DNA strand.

Such diagnostics are particularly useful for prenatal and even neonataltesting.

Point mutations and other sequence differences between the referencegene and “mutant” genes can be identified by other well-knowntechniques, such as direct DNA sequencing or single-strandconformational polymorphism, (see Orita et al., Genomics, 5, 874-879(1989)). For example, a sequencing primer may be used withdouble-stranded PCR product or a single-stranded template moleculegenerated by a modified PCR The sequence determination is performed byconventional procedures with radiolabelled nucleotides or by automaticsequencing procedures with fluorescent-tags. Cloned DNA segments mayalso be used as probes to detect specific DNA segments. The sensitivityof this method is greatly enhanced when combined with PCR. Further,point mutations and other sequence variations, such as polymorphisms,can be detected as described above, for example, through the use ofallele-specific oligonucleotides for PCR amplification of sequences thatdiffer by single nucleotides.

DNA sequence differences may also be detected by alterations in theelectrophoretic mobility of DNA fragments in gels, with car withoutdenaturing agents, or by direct DNA sequencing (for example, Myers etal., Science (1985) 230:1242) Sequence changes at specific locations mayalso be revealed by nuclease protection assays, such as RNase and SIprotection or the chemical cleavage method (see Cotton et at., Proc.Natl. Acad Sci USA (1985) 85: 43974401).

In addition to conventional gel electrophoresis and DNA sequencing,mutations such as microdeletions, aneuploidies, translocations,inversions, can also be detected by in site analysis (see, for example,Keller et al., DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA(1993)), that is, DNA or RNA sequences in cells can be analysed formutations without need for their isolation and/or immobilisation onto amembrane. Fluorescence in situ hybridization (FISH) is presently themost commonly applied method and numerous reviews of FISH have appeared(see, for example, Trachuck et al., Science, 250, 559-562 (1990), andTrask et al., Trends, Genet. 7, 149-154 (1991)).

In another embodiment of the invention, an array of oligonucleotideprobes comprising a nucleic acid molecule according to the invention canbe constructed to conduct efficient screening of genetic variants,mutation and polymorphisms. Array technology methods are well known andhave general applicability and can be used to address a variety ofquestions in molecular genetics including gene expression, geneticlinkage, and genetic variability (see for example: M. Chee et al.,Science (1996), Vol 274, pp 610-613).

In one embodiment, the array is prepared and used according to themethods described in PCT application WO95/11995 (Chee et al); Lockhart,D. J. et al. (1996) Nat. Biotech. L4: 1675-1680); and Schena, M. et al.(1996) Proc. Natl. Acad. Sci. 93: 10614-10619). Oligonucleotide pairsmay range from two to over one million. The oligomers are synthesized atdesignated areas on a substrate using a light-directed chemical process.The substrate may be paper, nylon or other type of membrane, filter,chip, glass slide or any other suitable solid support. In anotheraspect, an oligonucleotide may be synthesized on the surface of thesubstrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described is PCT application WO95/25116(Baldeschweiler et al.). In another aspect, a “gridded” array analogousto a dot (or slot) blot may be used to arrange and link cDNA fragmentsor oligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot b-lot or dot blot apparatus), materials (any suitablesolid support), and machines (including robotic instruments), and maycontain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any othernumber between two and over one million which lends itself to theefficient use of commercially-available instrumentation.

In addition to the methods discussed above, diseases may be diagnosed bymethods comprising determining, from a sample derived from a subject, anabnormally decreased or increased level of polypeptide or mRNA.Decreased or increased expression can be measured at the RNA level usingany of the methods well known in the art for the quantitation ofpolynucleotides, such as, for example, nucleic acid amplification, forinstance PCR, RT-PCR, RNase protection, Northern blotting and otherhybridization methods.

Assay techniques that can be used to determine levels of a polypeptideof the present invention in a sample derived from a host are well-knownto those of skill in the art and are discussed in some detail above(including radioimmunoassays, competitive-binding assays, Western Blotanalysis and ELISA assays). This aspect of the invention provides adiagnostic method which comprises the steps of: (a) contacting a ligandas described above with a biological sample under conditions suitablefor the formation of a ligand-polypeptide complex; and (b) detectingsaid complex.

Protocols such as ELISA, RIA, and FACS for measuring polypeptide levelsmay additionally provide a basis for diagnosing altered or abnormallevels of polypeptide expression. Normal or standard values forpolypeptide expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably humans, withantibody to the polypeptide under conditions suitable for complexformation The amount of standard complex formation may be quantified byvarious methods, such as by photometric means.

Antibodies which specifically bind to a polypeptide of the invention maybe used for the diagnosis of conditions or diseases characterized byexpression of the polypeptide, or in assays to monitor patients beingtreated with the polypeptides, nucleic acid molecules, ligands and othercompounds of the invention. Antibodies useful for diagnostic purposesmay be prepared in the same manner as those described above fortherapeutics. Diagnostic assays for the polypeptide include methods thatutilise the antibody and a label to detect the polypeptide in human bodyfluids or extracts of cells or tissues. The antibodies may be used withor without modification, and may be labelled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules known in the art may be used, several of which aredescribed above.

Quantities of polypeptide expressed in subject, control and diseasesamples from biopsied tissues are compared with the standard values.Deviation between standard and subject values establishes the parametersfor diagnosing disease. Diagnostic assays may be used to distinguishbetween absence, presence, and excess expression of polypeptide and tomonitor regulation of polypeptide levels during therapeuticintervention. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials or in monitoring the treatment of an individual patient.

A diagnostic kit of the present invention may comprise:

(a) a nucleic acid molecule of the present invention;(b) a polypeptide of the present invention; or(c) a ligand of the present invention.

In one aspect of the invention, a diagnostic kit may comprise a firstcontainer containing a nucleic acid probe that hybridises understringent conditions with a nucleic acid molecule according to theinvention; a second container containing primers useful for amplifyingthe nucleic acid molecule; and instructions for using the probe andprimers for facilitating the diagnosis of disease. The kit may fibercomprise a third container holding an agent for digesting unhybridizedRNA.

In an alternative aspect of the invention, a diagnostic kit may comprisean array of nucleic acid molecules, at least one of which may be anucleic acid molecule according to the invention.

To detect polypeptide according to the invention, a diagnostic kit raycomprise one or more antibodies that bind to a polypeptide according tothe invention; and a reagent useful for the detection of a bindingreaction between the antibody and the polypeptide.

Such kits will be of use in diagnosing a disease or susceptibility todisease in which members of the IL-8 like chemokine family areimplicated. Such diseases may include cell proliferative disorders,including neoplasm, melanoma, lung, colorectal, breast, pancreas, headand neck and other solid tumours; myeloproliferative disorders, such asleukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia,angiogenesis disorder, Kaposis' sarcoma; autoimmune/inflammatorydisorders, including allergy, inflammatory bowel disease, arthritis,psoriasis and respiratory tract inflammation, asthma, and organtransplant rejection; cardiovascular disorders, including hypertension,oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusioninjury, and ischemia; neurological disorders including central nervoussystem disease, Alzheimers disease, brain injury, amyotrophic lateralsclerosis, and pain; developmental disorders; metabolic disordersincluding diabetes mellitus, osteoporosis, and obesity, AIDS and renaldisease; infections including viral infection, bacterial infection,fungal infection and parasitic infection and other pathologicalconditions. Preferably, the diseases are those in which members of theIL-8 like chemokine family are implicated such as arthritis, rheumatoidarthritis (RA), psoriatic arthritis, osteoarthritis, systemic lupuserythematosus (SLE), systemic sclerosis, scleroderma, polymyositis,glomerulonephritis, fibrosis, lung fibrosis and inflammation, allergicor hypersensitivity diseases, dermatitis, asthma, chronic obstructivepulmonary disease (COPD), inflammatory bowel disease (IBD), Crohn'sdiseases, ulcerative colitis, multiple sclerosis, septic shock, HIVinfection, transplant rejection, wound healing, metastasis,endometriosis, hepatitis, liver fibrosis, cancer, analgesia, andvascular inflammation related to atherosclerosis. Such kits may also beused for the detection of reproductive disorders including infertility.

Various aspects and embodiments of the present invention will now bedescribed in more detail by way of example, with particular reference tothe INSP085 polypeptides.

It will be appreciated that modification of detail may be made withoutdeparting from the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Top ten results from BLAST against NCBI non-redundant databaseusing SEQ ID NO:8 (INSP085 full protein sequence).

FIG. 2: Alignment generated by BLAST between SEQ ID NO:8 (INSP085 fullprotein sequence) and the top five hits, small inducible cytokinesubfamily B member 15 (Mus musculus), K60 protein (Gallus gallus), sheepIL-8 precursor (Ovis aries), pig IL-8 precursor (Sus scrofa) and bovineIL-8 precursor (Bos taurus).

FIG. 3: Sig P cleavage site prediction for INSP085.

FIG. 4: Sig P peptide prediction for INSIP085.

FIG. 5: INSP085 coding exon organization in genomic DNA and position ofPCR primers.

FIG. 6: Nucleotide sequence and translation of cloned INSP085 ORF

FIG. 7: Map of pENTR-INSP085-6HIS

FIG. 8: Map of pEAK12d-INSP085-6HIS

EXAMPLES Example 1 INSP085 Protein BLAST Results

The INSP085 polypeptide sequence, shown in SEQ ID NO:8, was used as aBLAST query against the NCBI non-redundant sequence database. As can beseen in FIG. 1, the top hit is for an inducible cytokine (chemokineswere considered to be part of the cytokine family for a long time). Thethird to fifth hits are all for IL-8 precursors, thus providing flirterevidence that INSP085 is a member of the IL-8 like protein family.

Example 2 INSP085 Signal Sequence

FIGS. 3 and 4 show that INSP085 is predicted to possess a signal peptideat the start of the protein. As the data in FIG. 3 clearly shows, thesignal peptide cleavage site is thought to be between residues 16 and 17of the INSP085 full protein sequence. FIG. 4 shows that this sequence isnot an anchor peptide, so it is likely that INSP085 is a secretedprotein (0.818 probability) (Nielsen, H. et al 1997, ProteinEngineering, 10, 1-6; Nielsen, H., and Krogh, A.: Prediction of signalpeptides and signal anchors by a hidden Markov model. In Proceedings ofthe Sixth International Conference on Intelligent Systems for MolecularBiology (ISMB 6), AAAI Press, Menlo Park, Calif., pp. 122-130 (1998)).

Example 3 Cloning of INSP085 by Exon Assemble

1. PCR Amplification of Exons Encoding INSP085 from Genomic DNA.

PCR primers were designed to amplify exons 1, 2 and 3 of INSP085 (table1). The reverse primer for exon 1 (INSP085-exon1R) has an overlap of 18bases with exon 2 of INSP085 at its 5′ end. The forward primer for exon2 (INSP085-exon2F) has an 18 bp overlap with exon 1 of INSP085 at its 5′end. The reverse primer for exon 2 (INSP085-exon2R) has an overlap of 18bases with exon 3 at its 5′ end. The forward primer for exon 3(NSP085-exon3F) contains a 18 bp overlap with exon 2 at its 5′ end.

To generate exon 1 of INSP085, the PCR reaction was performed in a finalvolume of 100 μl and contained 1.5 μl of genomic DNA (0.1 μg/μl Clontechcat no. 65550-1), 2 μl of 10 mM dNTPs (Amersham Pharmacia Biotech), 6 μlof INSP085-exon1F (10 μM), 6 μl of INSP085-exon1R (10 μM), 5 μl of 10×Pfu buffer and 1 μl of Pfu polymerase (3 U/μl) (Promega cat. no. M774B).The PCR conditions were 94° C. for 2 min; 35 cycles of 9-4° C. for 30s,60° C. for 30s and 72° C. for 1 min; an additional elongation cycle of72° C. for S main; and a holding cycle of 4° C. Reaction products wereloaded onto a 1.5% agarose gel (1× TAE) and PCR products of the correctsize (158 bp) were gel-purified using a Qiaquick Gel Extraction Kit(Qiagen cat. no. 28704) and eluted in 50 μl of elution buffer (Qiag en).Exon 1 was subcloned into pCR4Blunt TOPO vector (Invitrogen) byincubating 4 μl of get purified PCR product, with 1 μl of salt solutionand 1 μl of topoisomerase modified pCR4 Blunt-TOPO vector.

The reaction mixture was incubated at RT for 30 mm. An aliquot of thisreaction (2 μl) was used to transform 50 μl of E. coli Top 10 multishotcells (Invitrogen) by heat shock as follows: cells and DNA were mixed ina 12 ml polypropylene tube. The mixture was stored on ice for 15 minthen heat shocked at 42° C. for exactly 30 sec. Samples were then storedon ice for a further 2 min, then diluted by addition of 250 μl of roomtemperature SOC medium and incubated for 1 h at 37° C. with shaking.Transformants (300 μl) were plated on LB plates containing 100 μg/ml ofampicillin and incubated over night at 37° C. Mini prep DNA was preparedfrom 5 ml cultures prepared from 30 of the resultant colonies using aQiaprep Turbo 9600 robotic system (Qiagen). Mini-prep DNA was eluted in50 μl of elution buffer. Plasmid mini prep DNA (200-500 ng) was thensubjected to DNA sequencing with T7 and T3 sequencing primers using theBigDyeTerminator system (Applied Biosystems cat. no. 4390246) accordingto the manufacturer's instructions. Sequencing reactions were purifiedusing Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates(Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems3700 sequencer. One of the clones containing the correct sequence ofexon 1 was then used as template for further amplification of exon 1 ina 50 μl PCR reaction containing 0.3 μl of miniprep DNA, 2 μl of 5 mMdNTPs (Amersham Pharmacia Biotech), 6 μl of INSP085-exon1F (10 μM), 6 μlof INSP085-exon1R (10 μM), 5 μl of 10× Pfu buffer and 0.5 μl of Pfupolymerase (3 U/μl) (Promega cat no. M774B). The PCR conditions were 94°C. for 2 min; 30 cycles of 94° C. for 30s, 60° C. for 30 s and 72° C.for 1 min; an additional elongation cycle of 72° C. for 3 min; and aholding cycle oaf 4° C. Reaction products were loaded onto a 1.5%agarose gel (1×TAE) and PCR products of the correct size (168 bp) weregel-purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704)and eluted in 30 μl of elution buffer (Qiagen).

To generate exon 2 of INSP085, the PCR reaction was performed in a finalvolume of 100 μl and contained 1.5 μl of human genomic DNA (0.1 μg/μl,Novagen cat. No. 69237), 2 μl of 10 mM dNTs (Amersham PharmaciaBiotech), 6 μl of INSP085-exon2F (10 μM), 6 μl of INSP085-exon2R (10μM), 5 μl of 10× Pwo buffer and 1 μl of Pwo polymerase (5 U/Ξl) (Roche,cat. No. 1 644 955). The PCR conditions were 94° C. for 2 min; 35 cyclesof 94° C. for 30s, 60° C. for 30s and 72° C. for 1 min; an additionalelongation cycle of 72° C. for 5 rain; and a holding cycle of 4° C.Reaction products were loaded onto a 1.5% agarose gel (1×TAE) and PCRproducts of the correct size (143 bp) were gel-purified using a QiaquickGel Extraction Kit (Qiagen cat. no. 28704) and eluted in 30 μl ofelution buffer (Qiagen).

To generate exon 3 of INSP085, the PCR reaction was performed in a finalvolume of 100 μl and contained 1.5 μl of human genomic DNA (0.1 μg/μl,Novagen cat. No 69237), 2 μl of 10 mM dNTPs (Amersham PharmaciaBiotech), 6 μl of INSP085-exon3F (10 μM), 6 μl of INSP085-exon3R (10μM), 5 μl of 10× Pwo buffer and 1 μl of Pwo polymerase (5 U/μl) (Roche,cat. No. 1 644 955). The PCR conditions were 94° C. for 2 min; 35 cyclesof 94° C. for 30s, 60° C. for 30s and 72° C. for 1 min; an additionalelongation cycle of 72° C. for 5 min; and a holding cycle of 4° C.Reaction products were loaded onto a 1.5% agarose gel (1×TAE) and PCRproducts of the correct size (131 bp) were gel-purified using a QiaquickGel Extraction Kit (Qiagen cat. no. 28704) and eluted in 30 μl ofelution buffer (Qiagen).

2. Assembly of Exons 1-3 to Generate the INSP085 ORF

Exons 1, 2 and 3 were assembled in a 50 μl PCR reaction containing 2 μlof gel purified exon 1, 5 μl of gel purified exon 2, 5 μl of gelpurified exon 3, 2 μl of 5 mM dNTPs, 64 μl of INSP085-EX1 (10 μM), 641of INSP085-EX2 (10 μM), 5 μl of 10× Pfu buffer, and 0.5 μl of Pfupolymerase (3 U/μl) (Promega). The INSP085-EX1 primer contains a partialattB1 site and Kozak sequence at the 5′ end. The INSP085-EX2 primercontains a 5 HIS sequence at its 5′ end. The reaction conditions were:94° C., 4 min; 10 cycles of 94° C. for 30s, 48° C. for 30s and 70° C.for 2 min; 25 cycles of 94° C. for 30s, 52° C., for 30s and 70° C. for 2min; an additional elongation step of 70° C. for 10 min; and a holdingcycle at 4° C. Reaction products were analysed on a 1.5% agarose gel(1×TAE). PCR products of the correct size (387 bp) were gel purifiedusing a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and elutedin 50 μl of elution buffer (Qiagen). The resultant PCR product containsthe ORF of INSP085.

3. Subcloning of the INSP085 ORF into pDONR201

The INSP085 ORF was subcloned into pDONR201 using the Gateway™ cloningsystem (Invitrogen). A partial attB1 recombination site was added to the5′ end of INSP085 ORF and a 6 HIS tag sequence, stop codon and attB2recombination site was added to the 3′ end of the INSP085 ORF in a 50 μlPCR reaction containing 2 μl of gel purified INSP085-ORF PCR product, 2μl of 5 mM dNTPs (Amersham Pharmacia Biotech), 6 μl of GCP-F (10 μM), 6μl of GCP-R (10 μM), 5 μl of 10× Pfu buffer and 0.5 μl of Pfu polymerase(5 U/μl) in a final volume of 50 μl. The PCR conditions wee 94° C. for 2min; 30 cycles of 94° C. for 30s; 55° C. for 30s and 72° C. for 1 min;an additional elongation step of 12° C. for 3 mm and a holding cycle of4° C. Reaction products were analysed on a 1.5% agarose gel (1×TAE) andPCR products of the correct size (445 bp, corresponding toGateway-modified NSP085 ORF) were gel purified using a Qiaquick GelExtraction Kit (Qiagen cat. no. 28704) and eluted in 50 μl of elutionbuffer (Qiagen). Gateway-modified INSP085 ORF was then transferred topDONR201 using BP clonase as follows: 5 μl of Gateway-modified INSP085ORF was incubated with 1.5 μl pDONR201 (0.1 μg/μl), 2 μl BP buffer and1.5 μl of BP clonase enzyme mix (Invitrogen) at RT for Oh. The reactionwas stopped by addition of 1 μl proteinase K (2 μg) and incubated at 37°C. for a further 10 min. An aliquot of this reaction (1 μl) was used totransform 20 μl of E. coli DH10B cells (Invitrogen) (diluted ⅕ insterile water) by electroporation using a Biorad Gene Pulser accordingto the manufacturer's recommendations. Electroporated cells weretransferred to 12 ml polypropylene tubes, diluted by addition of 900 μlof room temperature LB medium and incubated for 1 h at 37° C. withshaking. Transformants (100 μl) were plated on LB plates containing 40μg/ml of kanamycin and incubated cover night at 37° C. Mini prep DNA wasprepared from 5 ml overnight cultures from 8 of the resultant coloniesusing a Qiaprep Turbo 9600 robotic system (Qiagen). Mini-prep D>NA waseluted in 50 μl off elution buffer. Plasmid mini prep DNA (200-500 ng)was then subjected to DNA sequencing with pENTR-F and pENTR-R sequencingprimers using the BigDyeTerminator system (Applied Biosystems cat. no.4390246) according to the manufacturer's instructions. Sequencingreactions were purified using Dye-Es columns (Qiagen) or Montage SEQ 96cleanup plates (Millipore cat. no. LSKS09624) then analyzed on anApplied Biosystems 3700 sequencer.

4. Subcloning of the INSP085 ORF to Expression Vector pEAK12d

Plasmid eluate (1.5 μl) from a pDONR201 clot containing the correctsequence of the INSP085 ORF (pENTR-INSP085-6HIS, plasmid ID no. 13414,FIG. 4) was then used in a recombination reaction containing 1.5 μlpEAK1.2d vector (0.1 μg/μl), 20 μl LR buffer and 1.5 μl of LR clonase(Invitrogen) in a final volume of 10 μl. The mixture was incubated at RTfor 1 h, stopped by addition of 1 μl proteinase K (2 μg) and incubatedat 37° C. for a further 10 min. An aliquot of this reaction (1 μl) wasused to transform 20 μl of E. coli DH10B cells (Invitrogen) (diluted ⅕in sterile water) by electroporation using a Biorad Gene Pulseraccording to the manufacturer's recommendations. Electroporated cellswere transferred to 12 ml polypropylene tubes, diluted by addition of900 μl of room temperature SOC medium and incubated for 1 h at 37° C.with shaking. Transformants (100 μl) were plated on LB plates containing100 μg/ml of ampicillin and incubated at 37° C. overnight. Mini prep DNAwas prepared from 5 ml overnight cultures from 4 of the resultantcolonies using a Qiaprep Turbo 9600 robotic system (Qiagen) as describedabove. Mini-prep DNA was eluted in 100 μl of elution buffer. Plasmidmini prep DNA (200-500 ng) was then subjected to DNA sequencing withpEAK12-F and pEAK12-R sequencing primers using the BigDyeTerminatorsystem (Applied Biosystems cat no. 4390246) according to themanufacturer's instructions. Sequencing reactions were purified usingDye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat.no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.

CsCl gradient purified maxi-prep DNA was prepared from a 500 ml cultureof a sequence verified clone, pEAK12d-INSP085-6HIS (plasmid ID no.13413, FIG. 5) (Sambrook J. et al., in Molecular Cloning, a LaboratoryManual, 2^(nd) edition, 1989, Cold Spring Harbor Laboratory Press),resuspended at a concentration of 1 μg/μl in sterile water and stored at−20° C.

TABLE 1 Primers for INSP085 cloning and sequencing Primer Sequence(5′-3′) GCP Forward G GGG ACA AGT TTG TAC AAA AAA GCA GGC TTC GCC ACCGCP Reverse GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TCA ATG GTG ATG GTGATG GTG INSP085-exon1F ATG AGC TTG GCT ATC CTC ATA TGG T INSP085-exon1R

INSP085-exon2F

INSP085-exon2R

INSP085-exon3F

INSP085-exon3R TTA GCG TTT GTT ACC TGG GTT ATG AC INSP085-EX1 GCA GGCTTC GCC ACC ATG AGC TTG GCT ATC CTC AT INSP085-EX2 GTG ATG GTG ATG GTGGCG TTT GTT ACC TGG GTT AT pEAK12-F GCC AGC TTG GCA CTT GAT GT pEAK12-RGAT GGA GGT GGA CGT GTC AG pENTR-F TCG CGT TAA CGC TAG CAT GGA TCT CpENTR-R GTA ACA TCA GAG ATT TTG AGA CAC T7 TAA TAC GAC TCA CTA TAG GG T3CTC CCT TTA GTG AGG GTA ATT Underlined sequence = Kozak sequence Bold =Stop codon Italic sequence = His tag Highlighted sequence = overlap withadjacent exon

Example 4 Expression and Purification of INSP085

Further experiments may now be performed to determine the tissuedistribution and expression levels of the INSP085 polypeptides in vivo,on the basis of the nucleotide and amino acid sequence disclosed herein.

The presence of the transcripts for INSP085 may be investigated by PCRof cDNA from different human tissues. The INSP085 transcripts may bepresent at very low levels in the samples tested. Therefore, extremecare is needed in the design of experiments to establish the presence ofa transcript in various human tissues as a small amount of genomiccontamination in the RNA preparation will provide a false positiveresult. Thus, all RNA should be treated with DNAse prior to use forreverse transcription. In addition, for each tissue a control reactionmay be set up in which reverse transcription was not undertaken (a-ve RTcontrol).

For example, 1 μg of total RNA from each tissue may be used to generatecDNA using Multiscript reverse transcriptase (ABI) and random hexamerprimers. For each tissue, a control reaction is set up in which all theconstituents are added except the reverse transcriptase (-ve RTcontrol). PCR reactions are set up for each tissue on the reversetranscribed RNA samples and the minus RT controls. INSP085-specificprimers may readily be designed on the basis of the sequence informationprovided herein. The presence of a product of the correct molecularweight in the reverse transcribed sample together with the absence of aproduct in the minus RT control may be taken as evidence for thepresence of a transcript in that tissue. Any suitable cDNA libraries maybe used to screen for the INSP085 transcripts, not only those generatedas described above.

The tissue distribution pattern of the INSP085 polypeptides will providefurther useful information in relation to the function of thosepolypeptides.

In addition, further experiments may now be performed using thepEAK12d-INSP085-6HIS expression vectors. Transfection of mammalian celllines with these vectors may enable the high level expression of theINSP085 proteins and thus enable the continued investigation of thefunctional characteristics of the INSP085 polypeptides. The followingmaterial and methods are an example of those suitable in suchexperiments:

Cell Culture

Human Embryonic Kidney 293 cells expressing the Epstein-Barr virusNuclear Anti gen (WK293-EBNA, Invitrogen) are maintained in suspensionin Ex-cell VPRO serum-free medium (seed stock, maintenance medium, JRH).Sixteen to 20 hours prior to transfection (Day-1), cells are seeded in2× T225 flasks (50 ml per flask in DMEM/F12 (1:1) containing 2% FBSseeding medium (JRH) at a density of 2×10⁵ cells/ml). The next day(transfection day 0) transfection takes place using the JetPEI™ reagent(2 μl/μg of plasmid DNA, PolyPlus-transfection). For each flask,plasmid. DNA is co-transfected with GFP (fluorescent reporter gene) DNA.The transfection mix is then added to the 2×1225 flasks and incubated at37° C. (5% CO₂) for 6 days. Confirmation of positive transfection may becarried out by qualitative fluorescence examination at day 1 and day 6(Axio-vert 10 Zeiss).

On day 6 (harvest day), supernatants from the two flasks are pooled andcentrifuged e.g. 4° C., 400 g) and placed into a pot bearing a uniqueidentifier. One aliquot (500 μl) is kept for QC of the 6H is-taggedprotein (internal bioprocessing QC).

Scale-up batches may be produced by following the protocol called “PEItransfection of suspension cells”, referenced BP/PEI/HH/02/04, withPolyethyleneimine from Polysciences as transfection agent

Purification Process

The culture medium sample containing the recombinant protein with aC-terminal 6H is tag is diluted with cold buffer A (50 mM NaH₂PO₄; 600ml % NaCl; 8.7% (w/v) glycerol, pH 7.5). The sample is filtered thenthrough a sterile filter (Millipore) and kept at 4° C. in a sterilesquare media bottle (Nalgene).

The purification is performed at 4° C. on the VISION workstation(Applied Biosystems) connected to an automatic sample loader(Labomatic). The purification procedure is composed of two sequentialsteps, metal affinity chromatography on a Poros 20 MC (AppliedBiosystems) column charged with Ni ions (4.6×50 mm, 0.83 ml), followedby gel filtration on a Sephadex G-25 medium (Amersham Pharmacia) column(1.0×10 cm).

For the first chromatography step the metal affinity column isregenerated with 3 column volumes of EDTA solution (100M EDTA; 1M NaCl;pH 8.0), recharged with Ni ions through washing with 15 column volumesof a 100 mM NiSO₄ solution, washed with 10 column volumes of buffer A,followed by 7 column volumes of buffer B (50 mM NaH₂PO₄; 600 mM NaCl;8.7% (w/v) glycerol, 400 mM; imidazole, pH 7.5), and finallyequilibrated with 15 column volumes of buffer A containing 15 mMimidazole. The sample is transferred, by the Labomatic sample loader,into a 200 ml sample loop and subsequently charged onto the Ni metalaffinity column at a flow rate of 10 ml/min. The column is washed with12 column volumes of buffer A, followed by 28 column volumes of buffer Acontaining 20 mM imidazole. During the 20 mM imidazole wash looselyattached contaminating proteins are eluted from the column. Therecombinant His-tagged protein is finally eluted with 10 column volumesof buffer B at a flow rate of 2 ml/min, and the eluted protein iscollected.

For the second chromatography step, the Sephadex G-25 gel-filtrationcolumn is regenerated with 2 ml of buffer D (1.137M NaCl; 2.7 nm t KCl;1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; pH 7.2), and subsequently equilibrated with4 column volumes of buffer C (137 mM NaCl; 2.7 mM KCl; 1.5 mM KH₂PO₄; 8mM Na₂HPO₄; 20% (w/v) glycerol; pH 7.4). The peak fraction eluted fromthe Ni-column is automatically loaded onto the Sephadex G-25 columnthrough the integrated sample loader on the VISION and the protein iseluted with buffer C at a flow rate of 2 ml/min. The fraction wasfiltered through a sterile centrifugation filter (Millipore), frozen andstored at −80° C. An aliquot of the sample is analyzed on SDS-PAGE(4-12% NuPAGE gel; Novex) Western blot with anti-His antibodies. TheNuPAGE gel may be stained in a 0.1% Coomassie blue R250 stainingsolution (30% methanol, 10% acetic acid) at room temperature for 1 h andsubsequently destained in 20% methanol, 7.5% acetic acid until thebackground is clear and the protein bands clearly visible.

Foil owing the electrophoresis the proteins are electrotransferred fromthe gel to a nitrocellulose membrane. The membrane is blocked with 5%milk powder in buffer E (137 mM NaCl; 2.7 mM KCl; 1.5 mM KH₂PO₄; 8 mMNa₂HPO₄; 0.1% Tween 20, pH 7.4) for 1 h at room temperature, andsubsequently incubated with a mixture of 2 rabbit polyclonal anti-Hisantibodies (G-18 and H-15, 0.2 μg/ml each, Santa Cruz) in 2.5% milkpowder in buffer E overnight at 4° C. After a further 1 hour incubationat room temperature, the membrane is washed with buffer E (3×10 min),and then incubated with a secondary HRP-conjugated anti-rabbit antibody(DAKO, HRP 0399) diluted 1/3000 in buffer E containing 2.5% milk powderfor 2 hoes at room temperature. After washing with buffer E (3×10minutes), the membrane is developed with the ECL kit (AmershamPharmacia) for 1 min. The membrane is subsequently exposed to aHyperfilm (Amersham Pharmacia), the film developed and the western blotimage visually analysed.

For samples that showed detectable protein bands by Coomassie staining,the protein concentration may be determined using the BCA protein assaykit (Pierce) with bovine serum albumin as standard.

Furthermore, overexpression or knock-down of the expression of thepolypeptides in cell lines may be used to determine the effect ontranscriptional activation of the host cell genome. Dimerisationpartners, co-activators and co-repressors of the INSP085 polypeptide maybe identified by immunoprecipitation combined with Western blotting andimmunoprecipitation combined with mass spectroscopy.

1-2. (canceled)
 3. A purified nucleic acid molecule which encodes apolypeptide, wherein the polypeptide: (i) comprises the amino acidsequence as recited in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10 and/or SEQ ID NO:12; (ii) is a fragment thereof whichfunctions as a member of the IL-8 like chemokine family, or having anantigenic determinant in common with the polypeptide of (i): or (iii) isa functional equivalent of (i) or (ii).
 4. The purified nucleic acidmolecule according to claim 3, which comprises the nucleic acid sequenceas recited in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, and/or SEQ ID NO:11, or is a redundant equivalent or fragmentthereof; or wherein the nucleic acid molecule hybridizes under highstringency conditions with a nucleic acid molecule that encodes thepolypeptide.
 5. A vector comprising a nucleic acid molecule as recitedin claim
 3. 6. A host cell transformed with a vector according to claim5. 7-8. (canceled)
 9. A nucleic acid molecule according to claim 3, avector comprising a nucleic acid molecule according to claim 5, or ahost cell transformed with a vector according to claim 6, for use intherapy or diagnosis of disease. 10-12. (canceled)
 13. A pharmaceuticalor a vaccine composition comprising a nucleic acid molecule according toclaim 3, a vector comprising a nucleic acid molecule according to claim5, or a host cell transformed with a vector according to claim
 6. 14. Anucleic acid molecule according to claim 3, a vector comprising anucleic acid molecule according to claim 1 according to claim 5, or ahost cell transformed with a vector according to claim 6, or apharmaceutical composition comprising any of the above, for use in themanufacture of a medicament for the treatment of reproductive disorders,including infertility, cell proliferative disorders, including neoplasm,melanoma, lung, colorectal, breast, pancreas, head and neck and othersolid tumours; myeloproliferative disorders, such as leukemia,non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesisdisorder, Kaposis' sarcoma; autoimmune/inflammatory disorders, includingallergy, inflammatory bowel disease, arthritis, psoriasis andrespiratory tract inflammation, asthma, and organ transplant rejection;cardiovascular disorders, including hypertension, oedema, angina,atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, andischemia; neurological disorders including central nervous systemdisease, Alzheimer's disease, brain injury, amyotrophic lateralsclerosis, and pain; developmental disorders; metabolic disordersincluding diabetes mellitus, osteoporosis, and obesity, AIDS and renaldisease; infections including viral infection, bacterial infection,fungal infection, parasitic infection, rheumatoid arthritis (RA),psoriatic arthritis, osteoarthritis, systemic lupus erythematosus (SLE),systemic sclerosis, scleroderma, polymyositis, glomerulonephritis,fibrosis, lung fibrosis and inflammation, allergic, or hypersensitivitydiseases, dermatitis, asthma, chronic obstructive pulmonary disease,(COPD), Crohn's disease, ulcerative colitis, multiple sclerosis, septicshock, HIV infection, transplant rejection, wound healing, metastasis,endometriosis, hepatitis, liver fibrosis, cancer, analgesia, andvascular inflammation related to atherosclerosis, wherein said diseaseis a disease in which IL-8 like chemokines are implicated, and otherpathological conditions. 15-17. (canceled)
 18. A kit, wherein: a) thekit is useful for diagnosing disease comprising a first containercontaining a nucleic acid probe that hybridises under stringentconditions with a nucleic acid molecule according to claim 3; a secondcontainer containing primers useful for amplifying said nucleic acidmolecule; and instructions for using the probe and primers forfacilitating the diagnosis of disease, wherein the kit optionallyfurther comprises a third container holding an agent for digestingunhybridized RNA; or b) the kit comprises an array of nucleic acidmolecules, at least one of which is a nucleic acid molecule according toclaim
 3. 19-20. (canceled)